Sperm specific proteins

ABSTRACT

The present invention relates to sperm specific surface proteins, nucleic acid sequences encoding those proteins and antibodies raised against those proteins. Compositions comprising the sperm specific proteins or inhibitors of said proteins can be used in contraceptive applications.

[0001] This application claims priority under 35 U.S.C. §119(e) toprovisional patent application No. 60/176,885, filed Jan. 19, 2000.

US GOVERNMENT RIGHTS

[0002] This invention was made with United States Government supportunder Grant No. HD U54 29099, awarded by the National Institutes ofHealth. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The present invention is directed to sperm specific proteins thathave been isolated using 2-D gel analysis and microsequencing of theisolated proteins. These proteins are excellent candidates for use incontraceptive compositions, including contraceptive vaccinecompositions.

BACKGROUND OF THE INVENTION

[0004] Substantially continuous attention is focused on the developmentof improved contraceptive methods. One widely exploited technology isthe use of spermicides, essentially a chemical barrier that preventspenetration of sperm to the uterus or egg, or inhibits the activitythereof, thereby precluding fertilization. One of the most widely usedspermicides is a detergent, Nonoxynol-9. Reports indicate an increasedincidence of urogenital infections and cervicovaginal inflammation inwomen employing this detergent spermicide. McGroarty et al, Journal ofUrology, 152(3):831-833 (1994).

[0005] As an alternative to chemical detergents, authors have suggestedthe use of monoclonal antibodies as likely safe active agents fortopical applications, such as use in topical spermicides. See, e.g.,Cone et al, Am. J. Reprod. Immunol., 32:114-131 (1994). Studies concludethat in addition to the reduction or elimination of unwanted immunereactions, human monoclonal antibodies should present safe spermicidessince their dose and duration of application can be readily controlled,topical delivery minimizes systemic exposure and the monoclonal antibodycan be selected for safety and efficacy. Therefore, a sperm-activemonoclonal antibody delivered as a topical spermicide may producedesired anti-fertility effects without the negative side effectsaccompanied by detergent spermicides. See generally, Alexander,Scientific American, September:136-141 (1995). Accordingly, a goal inthe art continues to be the provision of a safe and effective spermicideemploying monoclonal antibodies.

[0006] Many investigators around the world are looking at thepossibility of the development of contraceptive vaccines based on spermantigens. See, e.g., Aitken et al, British Medical Journal, 49:88-99(1993), Freemerman et al, Biol. Reprod., 50:615-621 (1994) and Herr,Fertility Control, pp. 431-452 (Second Edition 1994). In thisconnection, work continues on human chorionic gonadotropin (hCG) as acontraceptive vaccine for women. Talwar, Current Opinion in Immunology,6:698-704 (1994) and European Patent 86304274.3. While clinical vaccinetrials are underway with this potential vaccine, the hCG immunogenemployed functions as an abortifactant, that is, immune responsesinduced by inoculation with this vaccine induce abortion of the earlyembryo or fetus. This may constitute an unacceptable form ofcontraceptive for many individuals.

[0007] As an alternative, a variety of sperm surface antigens have beenemployed in studies involving primate and rodent models. Thus, decreasedfertility rates resulted from the immunization of test animals withsperm surface antigens such as LDH-C4, O'Hern et al, Biol. Reprod.,52:331-339 (1995), PH-20, Primakoff et al, Nature, 335:543,546 (1988),RSA-1, O'Rand et al, J. Reprod. Immunol., 25:89-102 (1993) and fertilin,Ramarao et al, Mol. Reprod. Dev., 43:70-75 (1995). Disappointingly, inprimates, the highest rate of efficacy observed with a sperm antigen isabout 75 percent inhibition of fertility, O'Hern et al, supra. Thus todate there has not been identified a human sperm antigen that functionsas a contraceptive vaccine with a level of efficacy comparable to thatof oral contraception. Thus, it remains an object of those of skill inthe art to provide a safe and effective contraceptive vaccine with ahigh rate of fertility inhibition, on the order of the level of efficacygiven by oral contraceptives.

[0008] Additionally, because those receiving a contraceptive vaccinewill require periodic monitoring of serum antibody to determine if theyare “safe”, use of the sperm specific antigen as a target in assays tomeasure antibody concentration in persons receiving the vaccine isdesirable.

[0009] “Over the counter” assay or diagnostic kits for the detection ofhormones associated with pregnancy (hCG and others) have achievedwide-spread success in the marketplace, as an alternative or afirst-step to potentially embarrassing, inconvenient and expensivevisits to medical offices. In recent years, attention has been focusedon assays for the presence, and concentration of sperm in a usersejaculate. Both from the point of view of fertility counseling, as wellas clinical diagnosis in the case of rape, or for the purposes ofassaying for the presence and effectiveness of a vasectomy, a convenienttest kit, that could be safely and reliably employed at home, for thedetection of sperm in a sample, has become increasingly desirable. Inaccordance with the present invention, a test kit employing a monoclonalantibody against any of the sperm antigens disclosed herein, is alsowithin the scope of the present invention.

[0010] Purification of the sperm specific antigens is the first step inpreparation of an effective vaccine. The purified antigen, incorporatedin a pharmaceutically acceptable carrier, can be administered topatients desiring vaccination for contraception. Repeated vaccinationresults in the generation of antibodies against sperm, highly effectivein the binding of sperm. To monitor the development of an effectivelevel of antibodies, the purified antigen may be used as a test standardreagent, to determine the presence and amount of antibodies present inthe patient, through conventional diagnostics.

[0011] Definitions

[0012] In describing and claiming the invention, the followingterminology will be used in accordance with the definitions set forthbelow.

[0013] As used herein, “nucleic acid,” “DNA,” and similar terms alsoinclude nucleic acid analogs, i.e. analogs having other than aphosphodiester backbone. For example, the so-called “peptide nucleicacids,” which are known in the art and have peptide bonds instead ofphosphodiester bonds in the backbone, are considered within the scope ofthe present invention.

[0014] The term “peptide” encompasses a sequence of 3 or more aminoacids wherein the amino acids are naturally occurring or synthetic(non-naturally occurring) amino acids. Peptide mimetics include peptideshaving one or more of the following modifications:

[0015] 1. peptides wherein one or more of the peptidyl —C(O)NR— linkages(bonds) have been replaced by a non-peptidyl linkage such as a—CH₂-carbamate linkage (—CH₂OC(O)NR—), a phosphonate linkage, a—CH₂-sulfonamide (—CH 2—S(O)₂NR—) linkage, a urea (—NHC(O)NH—) linkage,a—CH₂-secondary amine linkage, or with an alkylated peptidyl linkage(—C(O)NR—) wherein R is C₁ C₄ alkyl;

[0016] 2. peptides wherein the N-terminus is derivatized to a —NRR₁group, to a —NRC(O)R group, to a —NRC(O)OR group, to a—NRS(O)₂R group,to a —NHC(O)NHR group where R and R₁ are hydrogen or C₁ C₄ alkyl withthe proviso that R and R₁ are not both hydrogen;

[0017] 3. peptides wherein the C terminus is derivatized to —C(O)R₂where R₂ is selected from the group consisting of C₁ C₄ alkoxy, and—NR₃R₄ where R₃ and R₄ are independently selected from the groupconsisting of hydrogen and C₁-C₄ alkyl.

[0018] Naturally occurring amino acid residues in peptides areabbreviated as recommended by the IUPAC-IUB Biochemical NomenclatureCommission as follows: Phenylalanine is Phe or F; Leucine is Leu or L;Isoleucine is Ile or I; Methionine is Met or M; Norleucine is Nle;Valine is Vat or V; Serine is Ser or S; Proline is Pro or P; Threonineis Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is Hisor H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K;Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys orC; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G,and X is any amino acid. Other naturally occurring amino acids include,by way of example, 4-hydroxyproline, 5-hydroxylysine, and the like.

[0019] Synthetic or non-naturally occurring amino acids refer to aminoacids which do not naturally occur in vivo but which, nevertheless, canbe incorporated into the peptide structures described herein. Theresulting “synthetic peptide” contain amino acids other than the 20naturally occurring, genetically encoded amino acids at one, two, ormore positions of the peptides. For instance, naphthylalanine can besubstituted for trytophan to facilitate synthesis. Other synthetic aminoacids that can be substituted into peptides include L-hydroxypropyl,L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl,beta.-amino acids, and isoquinolyl. D amino acids and non-naturallyoccurring synthetic amino acids can also be incorporated into thepeptides. Other derivatives include replacement of the naturallyoccurring side chains of the 20 genetically encoded amino acids (or anyL or D amino acid) with other side chains.

[0020] As used herein, the term “conservative amino acid substitution”are defined herein as exchanges within one of the following five groups:

[0021] I. Small aliphatic, nonpolar or slightly polar residues:

[0022] Ala, Ser, Thr, Pro, Gly;

[0023] II. Polar, negatively charged residues and their amides:

[0024] Asp, Asn, Glu, Gln;

[0025] III. Polar, positively charged residues:

[0026] His, Arg, Lys;

[0027] IV. Large, aliphatic, nonpolar residues:

[0028] Met Leu, Ile, Val, Cys

[0029] V. Large, aromatic residues:

[0030] Phe, Tyr, Trp

[0031] As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment.

[0032] As used herein, the term “C7/8 polypeptide” and like terms refersto polypeptides comprising SEQ ID NO: 2 and biologically activefragments thereof.

[0033] As used herein, the term “SAMP32 polypeptide” and like termsrefers to polypeptides comprising SEQ ID NO: 9 and biologically activefragments thereof.

[0034] As used herein, the term “C58 polypeptide” and like terms refersto polypeptides comprising SEQ ID NO: 16 and biologically activefragments thereof.

[0035] As used herein, the term “biologically active fragments” or“bioactive fragment” of an C7/8, SAMP32 or C58 polypeptide encompassesnatural or synthetic portions of those polypeptides that are capable ofspecific binding to at least one of the natural ligands of the nativepolypeptide.

[0036] “Operably linked” refers to a juxtaposition wherein thecomponents are configured so as to perform their usual function. Thus,control sequences or promoters operably linked to a coding sequence arecapable of effecting the expression of the coding sequence.

[0037] As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.

SUMMARY OF THE INVENTION

[0038] The present invention is directed to the isolation andcharacterization of novel testis and sperm-specific proteins that areexpressed on the surface of sperm. In particular the present inventiondescribes three sperm specific protein, C7/8, SAMP23, and C58. Theseproteins, and fragments thereof, are used in contraceptive vaccineformulations. The application is also directed to antibodies directedagainst these proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 is a copy of a multiple tissue Northern Blot, wherein C7/8cDNA was radiolabeled with P³² and hybridized to 2 ug poly-(A)+ mRNAs,revealing a message only in testicular RNA. Size of molecular weightmarkers is indicated at left, lanes 1-8 contain poly-(A)+ mRNA isolatedfrom spleen, thymus, prostate, testis, ovary, small intestinem colon andleucocyte, respectively.

[0040]FIG. 2 is a copy of a multiple tissue Northern Blot, whereinSAMP32 cDNA was radiolabeled with P³² and hybridized to 2 ug poly-(A)+mRNAs, revealing a message only in testicular RNA. Size of molecularweight markers is indicated at left, lanes 1-8 contain poly-(A)+ mRNAisolated from spleen, thymus, prostate, testis, ovary, small intestinemcolon and leucocyte, respectively. The lower panel of FIG. 2 shows theidentical blot probed with @-actin cDNA as a positive control.

[0041]FIG. 3 is an alignment of the C58 and Ly6D (E48 Antigen)_Humanamino acid sequences. The two amino acid sequences share 28.8% identityin 118 aa overlap region.

[0042]FIG. 4 is a copy of a multiple tissue Northern Blot, wherein C58cDNA was radiolabeled with P³² and hybridized to 2 ug poly-(A)+ mRNAs,revealing a message only in testicular RNA. Size of molecular weightmarkers is indicated at left, lanes 1-8 contain poly-(A)+ mRNA isolatedfrom spleen, thymus, prostate, testis, ovary, small intestinem colon andleucocyte, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention is directed to therapeutic and diagnosticmethods and compositions based on sperm specific surface proteins andthe nucleic acids encoding those proteins. In particular the presentinvention describes the isolation and characterization of three novelproteins, C7/8, SAMP23 and C58 that are expressed in human sperm cells.Antagonists of C7/8, SAMP23 and C58 polypeptide function can be used tointerfere with the binding and fusion of the sperm and the egg, and thussuch agents can be used as contraceptive agents. Furthermore, antibodiesagainst the C7/8, SAMP23 and C58 polypeptides can be used for thediagnosis of conditions or diseases characterized by expression oroverexpression of C7/8, SAMP23 or C58 polypeptides, or in assays tomonitor patients being treated with C7/8, SAMP23 or C58 agonists,antagonists or inhibitors.

[0044] In one embodiment, the present invention is directed to apurified polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 16 and aminoacid sequences that differs from SEQ ID NO: 2, SEQ ID NO: 9 or SEQ IDNO: 16 by one or more conservative amino acid substitutions. Morepreferably, the purified polypeptide comprises an amino acid sequencethat differs from SEQ ID NO: 2, SEQ ID NO: 9 or SEQ ID NO: 16 by 20 orless conservative amino acid substitutions, and more preferably by 10 orless conservative amino acid substitutions. Alternatively, thepolypeptide may comprise an amino acid sequence that differs from SEQ IDNO: 2, SEQ ID NO: 9 or SEQ ID NO: 16 by 1 to 5 alterations, wherein thealterations are independently selected from a single amino aciddeletion, insertion or substitution.

[0045] In one embodiment, the present invention provides methods ofscreening for agents, small molecules, or proteins that interact withC7/8, SAMP23 or C58. The invention encompasses both in vivo and in vitroassays to screen small molecules, compounds, recombinant proteins,peptides, nucleic acids, antibodies etc. which bind to or modulate theactivity of C7/8, SAMP23 or C58 and are thus useful as therapeutics ordiagnostic markers for fertility.

[0046] In one embodiment the C7/8 polypeptide, or bioactive fragmentsthereof, is used to isolate ligands that bind to the C7/8 polypeptideunder physiological conditions. The method comprises the steps ofcontacting the C7/8 polypeptide with a mixture of compounds underphysiological conditions, removing unbound and non-specifically boundmaterial, and isolating the compounds that remain bound to the C7/8polypeptides.

[0047] In another embodiment the SAMP23 polypeptide, or bioactivefragments thereof, is used to isolate ligands that bind to the SAMP23polypeptide under physiological conditions. The method comprises thesteps of contacting the SAMP23 polypeptide with a mixture of compoundsunder physiological conditions, removing unbound and non-specificallybound material, and isolating the compounds that remain bound to theSAMP23 polypeptides.

[0048] In one embodiment the C58 polypeptide, or bioactive fragmentsthereof, is used to isolate ligands that bind to the C58 polypeptideunder physiological conditions. The method comprises the steps ofcontacting the C58 polypeptide with a mixture of compounds underphysiological conditions, removing unbound and non-specifically boundmaterial, and isolating the compounds that remain bound to the C58polypeptides.

[0049] Typically, the C7/8, SAMP23 or C58 polypeptide will be bound to asolid support using standard techniques to allow rapid screeningcompounds. The solid support can be selected from any surface that hasbeen used to immobilize biological compounds and includes but is notlimited to polystyrene, agarose, silica or nitrocellulose. In oneembodiment the solid surface comprises functionalized silica or agarosebeads. Screening for such compounds can be accomplished using librariesof pharmaceutical agents and standard techniques known to the skilledpractitioner.

[0050] In accordance with one embodiment an antigenic composition isprovided wherein the composition comprises a polypeptide selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 16 andantigenic fragments of the polypeptides of SEQ ID NO: 2, SEQ ID NO: 9 orSEQ ID NO: 16. The compositions can be combined with a pharmaceuticallyacceptable carrier and administered to a patient to induce an immuneresponse. In one embodiment of the invention, antibodies directed to theC7/8, SAMP23 or C58 polypeptides, and fragments thereof, can beadministered to provide passive immunity and thereby provide acontraceptive effect.

[0051] The present invention encompasses vaccines useful forcontraception. In one aspect of the invention C7/8, SAMP23 or C58polypeptides, or antigenic fragments thereof, are delivered to a subjectto elicit an active immune response. The vaccine acts as a temporary andreversible antagonist of the function of the sperm surface proteins ofthe invention. For example, such vaccines could be used for activeimmunization of a subject, to raise an antibody response to temporarilyblock sperm's ability to bind or fuse to the egg.

[0052] In another aspect of the invention C7/8, SAMP23 or C58polypeptides, or antigenic fragments thereof, are used as vaccines forpermanent sterilization of a subject. Such vaccines can be used toelicit a T-cell mediated attack on the individuals sperm cells as amethod for irreversible sterilization. Methods for generating T-cellspecific responses, such as adoptive immunotherapy, are well known inthe art (see, for example, Vaccine Design, Michael F. Powell and Mark J.Newman Eds., Plenum Press, New York, 1995, pp 847-867). Such techniquesmay be particular useful for veterinary contraceptive or sterilizationpurposes, where a single dose vaccination may be desirable.

[0053] The preparation of vaccines containing an immunogenic polypeptideas the active ingredient is known to one skilled in the art (see, forexample, Vaccine Design, Michael F. Powell and Mark J. Newman Eds.,Plenum Press, New York, 1995, pp 821-902). The vaccines of the inventionmay be multivalent or univalent. Methods of introducing the vaccine mayinclude oral, intravaginal, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, and via scarification (scratchingthrough the top layers of skin, e.g., using a bifurcated needle) or anyother standard routes of immunization. The immunopotency of the C7/8,SAMP23 and C58 polypeptide antigens can be determined by monitoring theimmune response in test animals following immunization with the thoseprotein antigens, or by use of any immunoassay known in the art.Generation of a humoral (antibody) response and/or cell-mediatedimmunity, may be taken as an indication of an immune response. Testanimals may include mice, hamsters, dogs, cats, monkeys, rabbits,chimpanzees, etc., and eventually human subjects.

[0054] Suitable preparations of vaccines include injectables, either asliquid solutions or suspensions, however, solid forms suitable forsolution in, suspension in, liquid prior to injection, may also beprepared. The preparation may also be emulsified, or the polypeptidesencapsulated in liposomes. The active immunogenic ingredients are oftenmixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the vaccine preparationmay also include minor amounts of auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and/or adjuvants whichenhance the effectiveness of the vaccine.

[0055] Examples of adjuvants which may be effective, include, but arenot limited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.The effectiveness of an adjuvant may be determined by measuring theinduction of antibodies directed against an immunogenic polypeptidecontaining a C7/8, SAMP23 or C58 polypeptide epitope, the antibodiesresulting from administration of this polypeptide in vaccines which arealso comprised of the various adjuvants.

[0056] The polypeptides may be formulated into the vaccine as neutral orsalt forms. Pharmaceutically acceptable salts include the acid additionsalts (formed with free amino groups of the peptide) and which areformed with inorganic acids, such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,maleic, and the like. Salts formed with free carboxyl groups may also bederived from inorganic bases, such as, for example, sodium potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

[0057] The patient to which the vaccine is administered is preferably amammal, most preferably a human, but can also be a non-human animalincluding but not limited to cows, horses, sheep, pigs, fowl (e.g.,chickens), goats, cats, dogs, hamsters, mice and rats.

[0058] The vaccine formulations of the invention comprise an effectiveimmunizing amount of one or more of the sperm surface proteins of thepresent invention, and more particularly a polypeptide, or antigenicfragment thereof, selected from the group consisting of C7/8, SAMP23 andC58 polypeptides, and a pharmaceutically acceptable carrier orexcipient. Pharmaceutically acceptable carriers are well known in theart and include but are not limited to saline, buffered saline,dextrose, water, glycerol, sterile isotonic aqueous buffer, andcombinations thereof. One example of such an acceptable carrier is aphysiologically balanced culture medium containing one or morestabilizing agents such as stabilized, hydrolyzed proteins, lactose,etc. The carrier is preferably sterile. The formulation should suit themode of administration. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.

[0059] Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherethe composition is administered by injection, an ampoule of sterilediluent can be provided so that the ingredients may be mixed prior toadministration.

[0060] The precise dose of vaccine preparation to be employed in theformulation will also depend on the route of administration, and thenature of the patient, and should be decided according to the judgmentof the practitioner and each patient's circumstances according tostandard clinical techniques. An effective immunizing amount is thatamount sufficient to produce an immune response to the antigen in thehost to which the vaccine preparation is administered. Effective doses(immunizing amounts) of the vaccines of the invention may also beextrapolated from dose-response curves derived from animal model testsystems.

[0061] In instances where the recombinant antigen is a hapten, i.e., amolecule that is antigenic in that it can react selectively with cognateantibodies, but not immunogenic in that it cannot elicit an immuneresponse, the hapten may be covalently bound to a carrier or immunogenicmolecule; for instance, a large protein such as serum albumin willconfer immunogenicity to the hapten coupled to it. The hapten-carriermay be formulated for use as a vaccine.

[0062] The present invention thus provides a method of immunizing ananimal, comprising administering to the animal an effective immunizingdose of a vaccine of the present invention.

[0063] Another embodiment of the present invention comprises antibodiesthat are generated against an antigen selected from the C7/8, SAMP23 orC58 polypeptides. These antibodies can be formulated with standardcarriers and optionally labeled to prepare therapeutic or diagnosticcompositions. The antibodies generated against C7/8, SAMP23 or C58antigens have potential uses in vaccination against fertilization,sterilization, diagnostic immunoassays, passive immunotherapy, andgeneration of antiidiotypic antibodies.

[0064] Antibodies to the C7/8, SAMP23 and C58 polypeptides may begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric(i.e “humanized” antibodies), single chain (recombinant), Fab fragments,and fragments produced by a Fab expression library. These antibodies canbe used as diagnostic agents for the diagnosis of conditions or diseasescharacterized by expression or overexpression of the C7/8, SAMP23 orC58, or in assays to monitor patients being treated with C7/8, SAMP23 orC58 receptor agonists, antagonists or inhibitors. The antibodies usefulfor diagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. The antibodies may be used with orwithout modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule.

[0065] In one embodiment the antibodies generated against C7/8, SAMP23or C58 polypeptides are used in passive immunotherapy, in whichshort-term protection of a host is achieved by the administration ofpre-formed antibody directed against a heterologous organism. Theantibodies generated by the vaccine formulations of the presentinvention can also be used in the production of antiidiotypic antibody.The antiidiotypic antibody can then in turn be used for immunization, inorder to produce a subpopulation of antibodies that bind the initialantigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol.(Paris) 125c:373; Jerne, et al., 1982, EMBO J. 1:234).

[0066] In accordance with one embodiment an antibody is provided thatspecifically binds to a protein selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 16. In one preferredembodiment the antibody is a monoclonal antibody. Compositions can beprepared in accordance with the present invention comprising one or moreantibodies directed against epitopes present on C7/8, SAMP23 or C58 anda pharmaceutically acceptable carrier. In one embodiment the antibody isa monoclonal antibody.

[0067] The invention also encompasses antibodies, includinganti-idiotypic antibodies, antagonists and agonists, as well ascompounds or nucleotide constructs that inhibit expression of the C7/8,SAMP23 or C58 gene (transcription factor inhibitors, antisense andribozyme molecules, or gene or regulatory sequence replacementconstructs). The present invention also encompasses compositions thatcan be placed in contact with sperm cells to inhibit the function of theC7/8, SAMP23 or C58 protein (i.e. either by inhibiting the expression ofthe C7/8, SAMP23 or C58 protein or by interfering with the protein'sfunction). In particular the compositions may comprise peptide fragmentsof the respective C7/8, SAMP23 or C58 polypeptides, or analogs thereofthat are taken up by the sperm cells and compete for binding with thenative polypeptide's natural ligands. Such inhibitory peptides can bemodified to include fatty acid side chains to assist the peptides inpenetrating the sperm cell membrane. Compositions comprising an agentthat inhibits C7/8, SAMP23 or C58 functionality can be used to modulatefertility of an individual, and in one embodiment, the inhibitory agentsfunction as a male contraceptive pharmaceutical. In accordance with oneembodiment a composition is provided that comprises an eight to fifteenamino acid sequence that is identical to an eight to fifteen consecutiveamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 9 or SEQ ID NO: 16 and apharmaceutically acceptable carrier.

[0068] The present invention also encompasses nucleic acid sequencesthat encode the C7/8, SAMP23 or C58 polypeptide, as well as bioactivefragments and derivatives thereof. In particular, the present inventionis directed to nucleic acid sequences comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 14and SEQ ID NO: 15, or fragments thereof. In one embodiment, a purifiednucleic acid comprising at least 25 contiguous nucleotides that areidentical to any 25 contiguous nucleotides of SEQ ID NO: 1 are provided.In another embodiment, a purified nucleic acid comprising at least 25contiguous nucleotides that are identical to any 25 contiguousnucleotides of SEQ ID NO: 8 are provided. In another embodiment, apurified nucleic acid comprising at least 25 contiguous nucleotides thatare identical to any 25 contiguous nucleotides of SEQ ID NO: 14 areprovided. In other embodiments, the nucleic acid sequence comprises atleast 50 (contiguous) nucleotides, 100 nucleotides, 200 nucleotides, or500 nucleotides of a sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 8, SEQ ID NO: 14 and SEQ ID NO: 15.

[0069] The present invention also includes nucleic acids that hybridize(under conditions defined herein) to all or a portion of the nucleotidesequence represented by SEQ ID NO:1, SEQ ID NO: 8 or SEQ ID NO: 14 ortheir complement sequences. The hybridizing portion of the hybridizingnucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50)nucleotides in length. Hybridizing nucleic acids of the type describedherein can be used, for example, as a cloning probe, a primer (e.g., aPCR primer), or a diagnostic probe. It is anticipated that a DNAsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:8 or SEQ ID NO: 14, or fragments thereof can be used as probes to detectadditional members of the C7/8, SAMP23 or C586 families, respectively,and to detect homologous genes from other vertebrate species.

[0070] Nucleic acid duplex or hybrid stability is expressed as themelting temperature or Tm, which is the temperature at which a nucleicacid duplex dissociates into its component single stranded DNAs. Thismelting temperature is used to define the required stringencyconditions. Typically a 1% mismatch results in a 1° C. decrease in theTm, and the temperature of the final wash in the hybridization reactionis reduced accordingly (for example, if two sequences having >95%identity, the final wash temperature is decreased from the Tm by 5° C.).In practice, the change in Tm can be between 0.5° C. and 1.5° C. per 1%mismatch.

[0071] The present invention is directed to the nucleic acid sequencesof SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 14 and SEQ ID NO: 15 andnucleic acid sequences that hybridize to that sequence (or fragmentsthereof) under stringent or highly stringent conditions. In accordancewith the present invention highly stringent conditions are defined asconducting the hybridization and wash conditions at no lower than −5° C.Tm. Stringent conditions are defined as involve hybridizing at 68° C. in5×SSC/5× Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDSat 68° C. Moderately stringent conditions include hybridizing at 68° C.in 5×SSC/5× Denhardt's solution/1.0% SDS and washing in 3×SSC/0.1% SDSat 42° C. Additional guidance regarding such conditions is readilyavailable in the art, for example, by Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10.

[0072] In another embodiment of the present invention, nucleic acidsequences encoding the C7/8, SAMP23 or C58 polypeptide can be insertedinto expression vectors and used to transfect cells to expression ofthose respective proteins in the target cells. In accordance with oneembodiment, nucleic acid sequences encoding C7/8, SAMP23 or C58, or afragment or a derivative thereof, are inserted into a eukaryoticexpression vector in a manner that operably links the gene sequences tothe appropriate regulatory sequences, and the polypeptide is expressedin a eukaryotic host cell. Suitable eukaryotic host cells and vectorsare known to those skilled in the art. In particular, nucleic acidsequences encoding a C7/8, SAMP23 or C58 polypeptide may be added to acell or cells in vitro or in vivo using delivery mechanisms such asliposomes, viral based vectors, or microinjection. Accordingly, oneaspect of the present invention is directed to transgenic cell linesthat contain recombinant genes that express a polypeptide selected fromthe group consisting of C7/8, SAMP23 or C58.

[0073] In accordance with one embodiment the recombinant C7/8, SAMP23 orC58 polypeptides are be produced in large amounts and purified for usein vaccine preparations. Alternatively, the recombinant C7/8, SAMP23 orC58 polypeptides of the invention also have utility in immunoassays,e.g., to detect or measure in a sample of body fluid from a vaccinatedsubject the presence of antibodies to the antigen, and

[0074] thus to diagnose and/or to monitor immune response of the subjectsubsequent to vaccination.

[0075] Osteoglycin-Like Sperm Glycoprotein C7/8

[0076] Detection of Con-A binding proteins was carried out by exposing aWestern blot of 2-D electrophoretically separated total sperm proteinsto peroxidase-conjugated Con-A. Subsequently, the Con-A was visualizedby DAB counterstaining and demonstrated a number of positive regions at30, 40, 45, 75 and 90 kDa. More importantly, a region from 32 to 36 kDaand 5.0 to 5.0 pI that reacted positively was evident. The positivestaining with Con-A indicated then that this region of the 2-D containeda protein that was glycosylated and therefore was of possible secretoryor surface origin.

[0077] Western blots of electrophoretically 2-D separated sperm proteinsprobed with pools of fertile and infertile serum from 5 male and 5female individuals each were also examined. After incubation withperoxidase-conjugated anti-human IgG and counterstaining with DAB arepetoire of different proteins from the male and female infertileindividuals versus control seras from fertile males and females wasrevealed. A number of areas were highly immunogenic in the both the malefertile and infertile sera such as a broad pI-region around 90 kDa, aseries of proteins between 60 to 90 kDa of pI 5.5 to 6.0 as well asothers. Likewise, infertile female sera recognized some of these sameareas to varying degrees as well as others. Interestingly, a regionaround 34-38 kDa and 5.1 pI was found to be weakly immunogenic in malesera, indicating autoantigenicity, but was not recognized by either ofthe two fertile seras. Due to the remarkable correlation with theprevious Con-A results for this region it was decided to examine theproteins in this region further.

[0078] Two protein spots having relative molecular weights of 33 and 35kDa and estimated pIs of 5.0 and 5.1 respectively, were excised from aCoomassie stained 2-D gel. Microsequencing of peptides derived fromtryptic digestion of the protein spots were performed by both Edmandegradation and tandem mass spectrometry yielding a set of peptidesequences. A number of peptides from each protein spot were found toshared by both protein spots (Peptides 4, 6 and 7 of C7 corresponding topeptides 7, 9 and 10 of C8, respectively). Analysis of these proteinfragments by comparison to the non-redundant and EST NCBI databasesrevealed a few ESTs with significant matches to all three peptides foundin common to both proteins (Assession numbers: AA913806, AI33709,AI123225, AA432186, AI027115, AA782995, AA431165). Moreover, the 3peptides were found to be contained in the same open reading frame (ORF)in these ESTs lending support to the hypothesis that these peptides wereall part of some common protein. Further database analysis with theseESTs did not reveal any significant match to any known proteins andtherefore this protein was deemed to be new and unique and therefore ofinterest to our laboratory.

[0079] Oligonucleotides were manufactured to ends of the EST and a PCRreaction was performed with reverse transcribed human testicular cDNA. APCR product of the proper electrophoretic mobility was collected andsequenced before ³²P-labeling and used as a probe of a human testicularλDR2 cDNA library. After purification of several positive phage isolatesthe largest clone was sequenced in both directions. The nucleic acidsequence (SEQ ID NO: 1) of the cDNA library clone was 1337 bp in lengthand contained two start codons, both at the beginning of the samecontiguous ORF at bp 41 and at bp 59. Utilizing the methionine at bp 59as the start codon the ORF predicts a protein of 350 amino acids inlength (SEQ ID NO: 2) and 37.0 kDa in molecular weight with cleavage ofthe signal peptide and 39.6 kDa without this post-translationalmodification. Both of these predictions agree well with the region ofthe 2-D gel from which this protein spot was originally cored. A pI ofeither 5.4 or 5.44 was also predicted for the cleaved and uncleavedproteins respectively, likewise agreeing well with the originalpositioning on the 2-D gel system.

[0080] Further examination of the predicted protein revealed a consensusN-glycosylation site, NVSI, (Miletich and Broze, 1990) starting at aminoacid 134. The presence of a glycosylation site lends credence to theassumption that the proper protein spot was cloned. Numerous cAMP(starting at amino acid 119 and 347; Glass et al., 1983), PKC (startingat amino acids 66, 129, 182, 296, 339 and 346; Woodget et al., 1986) andcasein kinase2 (starting at amino acids 32, 85, 91, 188, 194, 200 and212; Pinna, 1990) consensus phosphorylation sites were also found.Finally, the original three peptide sequences used to identify the ESTwere present starting at amino acid positions 264-274, 310-317 and328-335. Another peptide microsequence was discovered at amino acids179-186 that was not included in the original EST translated ORF furtherreinforcing the conclusion that the proper cDNA clone was assigned tothe 2-D protein spot.

[0081] The complete ORF was subjected to analysis by database searching.Utilization of Blast identified a 29% identical and 49% homologous tomatch to a portion of murine osteoglycin (Ujita et al., 1995).Osteoglycin is a glycoprotein (Bentz et al., 1990) originally isolatedfrom bovine osteoblasts (Madisen et al., 1990), human osteocarcinomasources and vascular smooth muscle cells (Shanahan et al., 1997).Osteoglycin belongs to a larger family of leucine-rich-repeat proteinsthat include a number of extracellular matrix proteoglycans (Kajava,1998; Matsuuhima et al., 2000). The 68 amino acid region encompassingresidues 67-134 of murine osteoglycin (Ujita et al., 1995) aligns withamino acids 272-337 of the C-terminal region of C7/8. Notable in thisregion is a conserved serine residue that is implicated in covalentlinking of osteoglycin with extracellular matrix components. Likewise,the ProDomo algorithim (Altshul et al., 1990) identified a 50% homologyto the CDC24 family of guanine nucleotide releasing factors over the154-206 amino acid range of C7/8 and a 50% homology from amino acids274-304 to type II membrane binding proteins.

[0082] To ascertain the tissue specificity of the gene transcript aNorthern blot containing mRNA from various human tissues was probed withthe radiolabeled C7/8 cDNA (FIG. 1). A single prominent band ofapproximately 1.5 Kb was observed only in the lane containing testicularmRNA. The size of the mRNA transcript in the autoradiogram also verifiedthat, in all probability, a near full-length cDNA had been isolated.Probing the blot with radiolabeled β-actin (not shown) was performed asa quality control for the mRNA on the membrane. As opposed to theseresults with a sperm-specific osteoglycin-like cDNA bovine osteoglycincDNA applied to Northern blots reveals 2 transcripts of 1.9 and 2.4 Kbin size and is restricted to cells of the bone lineage (Madisen et al.,1990). An array of 76 different human tissue mRNAs were then hybridizedto a radiolabeled C7/8 on a dot-blot to examine a larger repetoire oftissues. The results confirmed that the C7/8 is indeed transcribed inadult testis.

[0083] In order localize and characterize human C7/8 (h-C7/8) amonospecific polyclonal sera was generated for immunological studies.The h-C7/8 ORF was expressed in E. coli using a pET28 plasmid (Novagen).Oligonucleotides bracketing the ORF were designed and the cloning wascarried out as described in Example 1. Induction of the plasmid-bearingE. coli with 1 mM IPTG in a 20 ml shake-flask was performed at anapproximate OD₆₀₀ of 0.7 and 1.0 OD-ml samples were removed at 1 hrintervals for 3 hrs. The culture expressing h-C7/8 doubled in OD₆₀₀during the 3 hr time period as opposed to a 30 min doubling time beforeinduction.

[0084] Purified rec-h-C7/8 was utilized to generate a monospecificpolyclonal antisera in virgin female Lewis rats. Western blotting 1-Dgels was employed to test the antisera generated against the recombinantprotein. The primary and secondary antibodies alone (lanes 1 and 2,respectively) failed to recognize rec-h-tekB1 whereas primary andsecondary antibodies together reacted with the recombinant immunogen.When an extract of human sperm proteins was separated on 2-D gels,transferred to a nitrocellulose membrane and sequentially Gold- andimmuno-stained with rat-rec-h-tekB1 three h-tekB1 isoforms werespecifically immunostained. These three h-tekB 1 isoforms migrated at aidentical moleular weight (53.5 kDa) but varied slightly in charge withpI's from 5.25 to 5.35. This observation indicates that there are threedifferently modified forms of the h-tekB1 present in human sperm.

[0085] The rat α-rec-h-C7/8 sera was utilized to examine theimmunohistochemical localization of the C7/8 protein within human sperm.Live staining of human sperm was negative indicating that the C7/8protein was not located in an accessible region of the human sperm.However, when mounted and permeabilized human sperm were probed with therat α-rec-h-C7/8 sera, counter-stained and examined a different resultwas obtained. Preimmune sera used as a control demonstrated nofluorescence while the immune sera reacted positively with a bandedregion of the human sperm localized to the base of the acrosome. Thispattern of staining was consistent in all the sperm observed althoughoccasional sperm demonstrated a punctate granular localization. In alllikelihood the localization of the human C7/8 protein is in theequitorial segment of the human sperm.

[0086] To definitively prove the localization of the human C7/8 proteinhuman sperm were subjected to electronmicroscopic examination. Stainingwas performed with the rat α-rec-h-C7/8 sera, and data showed that theimmunogold beads are predominantly present within the equitorial segmentof the human sperm. The equitoral segment of the human sperm is aspecialized region of the acrosome known previously to be involved withmembrane fusion events of the sperm with the egg as opposed to purelybinding events. Accordingly it is anticipated that the C7/8 protein mayparticipate in sperm binding and fusion with the egg. Thereforecompounds that interfere with C7/8 function will provide effectivecontraceptive agents.

[0087] Sperm Acrosomal Membrane Protein 32 (SAMP32)

[0088] Sperm Acrosomal Membrane Protein 32 (SAMP32) was initiallyisolated from 2-D gels and was initially designated “C71”. Inparticular, human sperm proteins, partitioned into the Triton X-114detergent phase, were resolved by isoelectric focusing (IEF) in thefirst dimension, and by SDS-PAGE in the second dimension. Protein spotsfrom the Commassie-stained gels were then cored for sequencing by massspectrometry. Among the several dozens of sperm proteins sequenced werefour spots named C71, 72, 74, and 75. These spots shared the samepeptide sequence according to the mass-spectrometry analysis(ASTPEVQSEQSSVR, SEQ ID NO: 7, was the longest). Interestingly however,their apparent molecular weights varied from 30 to 34 kD and their pIsranged from 4.5 to 5.5. Vectorial labeling showed that at least one ofthe four spots (C74) could be labeled by using biotin.

[0089] When the peptide sequence of SEQ ID NO: 7 was used in databasesearch, no known genes were found in the non-redundant protein databasesuggesting that it is a novel protein. There were, however, two exactmatches from the expressed sequence tag database (Entry AI419884 andAI809484) which overlap each other. Two oligo DNA primers, based onAI419884, were then designed to amplify cDNA from the human testis cDNAlibrary by PCR, which was followed by confirming DNA sequencing. Byscreening the lambda DR2 human testis cDNA library with the cDNAfragment as probe, 16 clones were picked up in the secondary round. Ofthese, clone 1-4-1 (1050 bp) had the largest size. Subsequent DNAsequencing confirmed that AI419884 was embedded in it. A 3′RACE was doneto amplify the full length clone (1455) as the expressed sequence tag(EST) database search and northern blot suggested a short of 400 bp in3′UTR of clone 1-4-1. The full-length clone is 1455 bp nucleotide long(SEQ ID NO: 8) and contains a contiguous open reading frame of 294 aminoacids (SEQ ID NO: 9). The predicted molecular weight and pI are 32 kDand 4.57 respectively. A FASTA search against the current database,using the full-length open reading frame, revealed homology to two genesfrom different species. They are malaria Circumsporozoite protein CSPand fission yeast KRP I.

[0090] Circumsporozoite protein is the immunodominant surface antigen onthe sporozoite. The sporozoite is the infective stage of the malariaparasite that is transmitted from the mosquito to the vertebrate host.CSP consists of 358 amino acids, the first 19 amino acids contain asignal peptide and amino acids 98-277 contain repeat that are thought tobe important in anchoring the protein to membranes. There repeat unitsare not found in SAMP32. The amino terminus of SAMP32 shares 30.088%identity and 53.09% similarity in a 113 amino acid region (amino acids22-131 of SAMP32 compared to 259-366 of CSP).

[0091] Dibasic Processing Endoprotease Precursor (KRP 1) is required forcell vaibility in fission yeast. It is a subtilisin-like serine proteasethat cleaves P-factor precursor and other precusors. This proteinbelongs to the type I membrane proteins and to peptidase family S8. Thecarboxy terminus of SAMP32 shares 25.309% identity and 51.85% similarityin a 162 amino acid region (amino acids 123-263 of SAMP32 compared to551-707 of KRP1).

[0092] SAMP32 is Phosphorylated In Vivo and is a Predicted Transmembrane

[0093] By running the algorithms provided in the PredictProtein program,three possible sites for phosphorylation by Casein Kinase II weredisclosed. Of these, at lease one, i.e. the serine residue at amino acidposition 256, was shown to be phosphorylated in vivo by massspectrometry analysis of peptide sequences. In addition, the proteinarchitecture was analyzed with algorithms provided in the Simple ModularArchitecture Research Tool on the ExPASy server. This analysis revealeda signal peptide from amino acid 1 to 29, a low complexity region fromamino acid 39 to 61 and a transmembrane domain from amino-acid 222 to242. The low complexity region is comprised mainly of glutamic acid(35%). In addition, prediction indicated possible N-Glycosylation ofthree amino acid residue(s) at aa31, 54, and 155.

[0094] Expression of SAMP32 is Exclusively Testis

[0095] To examine the expression of SAMP32 in various tissues, amulti-tissue northern blot was done first. A DNA fragment containing theentire ORF of SAMP32 was used as a probe. As shown in FIG. 2, SAMP32 wasonly detected in testis in a total of 8 tissues including spleen,thymus, prostate, ovary, small intestine, colon and peripherallymphocytes, which suggested that SAMP32 is testis specific. Thisspecificity was further confirmed by RNA dot blot hybridization. In thisexperiment, ³²P labeled SAMP32 was used to probe a membrane dotted withRNA samples extracted from 67 human tissues. In agreement with themulti-tissue northern blot result, only testis yield a hybridizationsignal.

[0096] When the entire cDNA sequence of SAMP32 was searched against thehuman genomic database, SAMP32 was localized to 6q15-16.2 of chromosome6 (Genebank entry, AL136096). Comparison of the cDNA sequence with thecorresponding genomic sequence revealed that SAMP32 consists of a totalof 7 exons ranging from 60 bp to 650 bp long. They were distributedacross a 19 kb region on chromosome 6. In all cases, the nearlyinvariant splicing consensus GU and AG in the 5 prime and 3 prime end ofthe intron have been found.

[0097] To study the SAMP32 gene expression at the protein level,antiserum against recombinant SMARC32 was generated. Part of the openreading frame of SAMP32 was amplified by PCR, inserted into pET28bexpression vector and expressed in E. coli. Purified protein as a singleband on SDS-PAGE was used to inject the rats. Antiserum produced wasused to blot human total sperm protein extracted with TritonX114. Theantiserum detected mainly three bands with apparent molecular weights of54, 48, and 41 Kd respectively and the 48 Kd band was the most abundantone quantitatively. The size of the molecular weight and the result ofthe western blot using the recombinant protein indicated that the highmolecular weight bands are likely dimers. To determine if this antiserumrecognizes protein spots that were cored from the two-dimensional gel,proteins that were extracted with TritonX-114 and partitioned intoTritonX-114, were loaded onto a two dimensional gel. After transfer tonitrocellulose membrane, protein spots were detected using the sameantiserum as described above.

[0098] Considering the hypothesis that SAMP32 is a putativetransmembrane phosphoprotein, the antibody agains SAMP32 was used tolocalized its position on human spermatozoa. The antiserum stained thewhole acrosomal cap and the equatorial bar of human spernatozoa inimmunofluorescence staining. The same antiserum stained mainly the innerand the outer acrosomal membrane alone with some staining of matrix,which strongly suggested that SAMP32 is acrosome-associated proteinlocated in the sperm acrosome, hence the name Sperm AcrosomeMembrane-associated Protein (SAMP32).

[0099] To follow SAMP32 expression in-situ during acrosomal development,human testis were from clinical patients. The tissue was digested withtrypsin, micrococcal nuclease and collagenase. The dissociated cellsmear on slides were then stained with rat anti-rSMARC antiserum. Thesperm nuclei were counter-stained with DAPI at the same time tofacilitate localization. SAMP32 was present in all stages of acrosomedevelopment including the Golgi phase, the cap phase in the roundspermatids, the acrosomes of elongating spermatids as well as theelongated mature sperm. The patterns are just like those found in SP10,a known acrosomal associated molecule.

[0100] To determine the fate of SAMP32 after the acrosomal reactionfresh sperm were washed and acrosomal reactions were induced byincubation progesterone. The acrosomal reacted sperm were stained firstusing SAMP32 as primary antibody and TRITC-conjugated donkey anti-rat Igas secondary antibody. This was followed by staining with Con-Aconjugated with FITC. By staining SAMP32 in acrosomal reacted sperm itwas shown that SAMP32 is still associated with equatorial segmentfollowing capacitation and acrosomal reaction. Thus it is anticipatedthat SAMP32 plays an important role in sperm egg interaction and thus inan appropriate immunological target for contraceptive vaccines.

[0101] Sperm Membrane Protein C58

[0102] Differential solubilization and phase partitioning using TX-114followed by 2-D gel electrophoresis was used to identify a novel,hydrophobic, putative human sperm membrane protein, which was named C58.The cDNA of the protein was cloned using a human testis EST. Notablefeatures of C58 are: 1) a 19 aa amino terminal signal peptide, 2) aLy-6/urokinase plasminogen activator receptor like domain (aa 22-112),3) a potential transmembrane domain near the carboxy terminus (aa101-124), and 4) a carboxy terminal cleavage site for transamidase, (aa97-99) suggesting C58 is glycosylphosphatidylinositol (GPI) anchored.Transcripts for C58 were expressed only in testis as studied by Northernblot of 8 human tissues and by dot blot analysis of 76 human tissueRNAs.

[0103] Recombinant C58 was produced in E. coli using the pET 28b vector.Antibody against the recombinant protein was successfully generated inrats. The antibody reacted with a sperm protein at an expected molecularweight of 12.5 kDa on 1-D gels and recognized the native 2-D spot thatwas initially cored for microsequencing C58. Preliminary experiments onthe indirect immunofluorescence localization of the protein revealedthat it is on the entire surface of the human sperm. The bioinformaticstudy of the c-DNA derived amino acid sequence revealed that it is aputative GPI anchored protein belonging to the Ly-6 family.

[0104] The location of C58 on the entire surface of the human sperm andthe fact that the protein is expressed only in sperm make this proteinan ideal target for contraceptive agents. In accordance with oneembodiment an antigenic composition is provided comprising an antigenselected from the group consisting of SEQ ID NO: 16 or antigenicfragments thereof. In addition, compositions comprising a nucleic acidcomprising the nucleic acid sequence of SEQ ID NO: 18 are also provided.

EXAMPLE 1 Isolation and Characterization of C7/8

[0105] 2-D Electrophoresis, Standard Protein Gels and Western Blotting

[0106] Human sperm proteins for 2-D gel electrophoresis were solubilizedand separated as previously described by Naaby-Hansen et al. (1997).Briefly, fresh semen specimens were allowed to liquify for up to 3 hbefore centrifugation on two-layer (80% and 55% in Ham s F-10 medium)Percoll density gradients. After subsequent washing of the sperm pelletcollected from the bottom of the 80% gradient 3 times in Ham s F-10medium, the sperm were solubilized in a lysis buffer consisting of 2%(v:v) NP-40, 9.8 M urea, 100 mM DTT, 2% ampholines (pH 3.5-10) and acocktail of protease inhibitors before performing isoelectric focusingon 0.15 mg of sperm proteins in a 15×0.15 cm acrylamide rod containing acarrier ampholine (Pharmacia) composition of 28% pH 3.5-5, 20% pH 5-7,7% pH 7-9 and 45% pH 3.5-10 in a gel composition described previously(Naaby-Hansen et al., 1997). Prior to electrophoresis the tubescontaining the sperm protein extract were overlayed with a buffercontaining 5% NP-40, 1% ampholines (pH 3.5-10), 8 M urea and 100 mM DTT.Isoelectric focusing was conducted in steps: 2 h at 200 V, 5 h at 500 V,4 h at 800 V, 6 h at 1200 V and 3 h at 2000 V. Second dimensionalelectrophoresis was performed by laying the isoelectrically focused tubegel onto a 0.15×20 cm linear (9-15% acrylamide) slab gel in a Protean IIxi Multi-Cell apparatus (Bio-Rad).

[0107] Standard SDS-PAGE gel electrophoresis (Coligan et al., 1995) forrecombinant proteins and prokaryotic cell lysates were performed on16×18 cm gel electrophoresis apparatus (BioRad) with 0.75 mm spacerswith 12% polyacrylamide separating gels. Protein or cellular sampleswere either suspended in standard Laemmli Buffer and boiled for 10 minbefore addition to the gel or treated with iodoacetic acid by theprocedure of Crestfield et al. (1963). Standard E. coli lysatesconsisted of 1 OD-ml of bacterial culture, pelleted before suspension inloading buffer.

[0108] Electrophoretic transfer to nitrocellulose membranes of separatedproteins was performed in Transblot Buffer (25 mM Tris (pH 8.3), 192 mMGlycine and 20% Methanol) for 1 A-Hr. The membranes were either utilizedintact for 2-D gels or cut into strips for standard protein gels,blocked with 5% fat-free dry milk in PBS-Tween (1×PBS (pH 7.4), 0.05%Tween) and incubated with either rat antisera or pools of fertile orinfertile sera from 5 individuals in blocking buffer for 1 hr.Immunodetection was performed with horseradish peroxidase-conjugatedgoat anti-rat IgG (Jackson ImmunoResearch) at a 1:5000 dilution inblocking buffer and visualized with diaminobenzidine (Sigma) in H₂O₂ orTMB (KPL).

[0109] Screening of the 2-D blot with Con-A was performed by incubatingWestern transferred 2-D gels of human sperm proteins first with TBS (10mM Tris-HC1 (pH 8.0), 150 mM NaCl) then with TBS containing 1% gelatin(TBSG) for 1 hr (Auer et al., 1995). After rinsing in TBS, the membranewas incubated with peroxidase-conjugated Con-A (5 μg/ml) in TBSGcontaining 1 mM MnCl₂ and 1 mM CaCl₂ cations for 1 hr at roomtemperature before rinsing briefly twice with TBSG. After washing themembrane 4 times for 15 min each with TBSG/cations staining wasperformed with diaminobenzidine (Sigma) in H₂O₂. Screening of Westernblots with 2-D separated human proteins with fertile and infertile serawas performed by blocking and washing as described above prior toincubation with pools of 5 individual's sera.

[0110] Protein Microsequencing

[0111] Sequencing was performed by the W. M. Keck Foundation Center forBiomedical Mass Spectrometry. Briefly, protein spots were cut from thepolyacrylamide gel as closely as possible, minced, washed and dried in50% methanol before dehydration in acetonitrile and rehydration in 50 μlof 10 mM dithiothreitol in 0.1 M ammonium bicarbonate for 1 hr at 55° C.The sample was alkylated in 50 μl 50 mM iodoacetamide and 0.1M ammoniumbicarbonate in the dark for 1 hr at room temperature before 2 rounds ofwashing with 0.1 M ammonium bicarbonate and dehydration in acetonitrileprior to vacuum dessication and treatment with 12.5 ng/μl trypsin in 50mM ammonium bicarbonate on ice for min. After removal of any excesstrypsin solution 20 μl of ammonium bicarbonate was added and the samplewas digested overnight at 37° C. before extraction of the peptides in50% acetonitrile/5% formic acid and analysis LC-MS analysis. Edmandegradation was performed on a PE Biosystems Model 494 according tomanufacturer's instructions.

[0112] RT-PCR

[0113] Oligonucleotides designed from the EST and cDNA library cloneswere manufactured by GibcoBRL. Oligonucleotides (EST forward primer:5′-CTTGCTCTAGCAGCAGCAGAAC-3′ (SEQ ID NO: 3); EST reverse primer:5′-TCATAACACATGACACATAAAGATGTTGGC-3′(SEQ ID NO: 4)) to the EST(AA913806) were utilized to generate a 430 bp probe by reversetranscription of human testicular RNA (Clontech). PCR reactions wasperformed by reverse transcribing 0.05 μg poly-(A)+ RNA in a 20 μlreaction by combining 0.5 μg oligo-d(T)₁₂₋₁₈ RNA anddiethylpyrocarbonate (DEPC)-treated H₂O prior to heating to 65° C. for 5min. Subsequently, 2 μl 10×RT buffer, 0.5 μl placental ribonucleaseinhibitor (36 U/μl; Promega), 1 μl 10 mM 4dNTPs and 1 μl AMV reversetranscriptase (23 U/μl; Stratagene) were added, vortexed and the mixturewas incubated for 60 min at 42° C. After the addition of 80 μlDEPC-treated H₂O, 1 μl aliquots of the cDNA solution were amplified for40 cycles with denaturation at 94° C., annealing at 60° C. for 30 secand polymerization at 68° C. for 3 min, as specified by the recombinantThermophilus thermophilus (rTth) polymerase manufacturer (Perkin Elmer).Separation and isolation of the PCR products was achieved byelectrophoresis of reaction aliquots in 1.5% agarose gels made 1× in TAE(40 mM Tris-acetate, 1 mM EDTA) buffer followed by ethidium bromidestaining, UV visualization and photography. Collection of specificRT-PCR fragments was performed by electroelution from the agarose gelfollowed by precipitation, quantitation on agarose gels versus standardsand ligation into a pCR2.1-TOPO cloning vector according tomanufacturer's instructions (InVitrogen). Sequencing was performed bythe University of Virginia Biomolecular Research Facility.

[0114] Screening of cDNA Library and Northern Blotting

[0115] The sequenced 430 bp insert obtained by RT-PCR was purified byEco RI-treating the pCR2.1-TOPO cloning vector and collecting thereleased cDNA fragments by band-excision after agarose gel separation.Fifty ng of the purified fragment was denatured by boiling thenradiolabeled with (α-³²P)-dCTP by including the two degenerateoligonucleotides described above in the random priming procedure ofFeinberg and Vogelstein (1984). The radiolabeled fragment was purifiedon an Elutip-D column (Schleicher and Schuell) and hybridized to six 137mm plaque lifts (Magna Nylon Transfer Membranes, MSI) containing a totalof 240,000 phage from a human testicular λDR2 5′-stretch cDNA library(Clontech) in a solution containing 50% Formamide, 5×SSC, 5× Denhardt sSolution, 0.25 μg/ml yeast RNA, 0.5% SDS and 0.05 M sodium phosphate (pH7.0) at 42° C. After overnight hybridization the filters were washed ina final solution of 0.2×SSC/0.2% SDS at 52° C. before mounting, exposureto XAR-5 film (Kodak) and development. Twenty primary isolates wererescreened twice and the remaining 8 positives were converted from λDR2to pDR2 according to manufacturer's instructions in AM1 cells.Sequencing was performed by the University of Virginia BiomolecularResearch Facility in both directions and the nucleotide and amino aciddata were analyzed using the Genetics Computer Group and SEQWeb(Madison, Wis.) program packages.

[0116] To generate a probe for tissue specificity analysis 50 ng of thepurified full-length 1337 bp C7/8 cDNA was radiolabeled as describedabove except for the omission of the EST oligonucleotides. Theradiolabeled cDNA was isolated and hybridized in ExpressHyb (Clontech)to either a Human Multiple Tissue Northern (Clontech) or a Human RNAMaster Blot (Clontech). All prehybridizations, hybridizations andwashings were performed according to manufacturer s instructions. TheHuman Multiple Tissue Northern contained 2 μg of poly-(A)⁺ RNA fromspleen, thymus, prostate, testis, ovary, small intestine, mucosal liningof the colon and peripheral blood leukocytes per lane while the HumanRNA Master Blot contained various amounts (100-500 ng) of poly-(A)⁺ RNAsfrom 76 different tissues normalized to various housekeeping genes.

[0117] Expression and Isolation of Recombinant Human C7/8

[0118] The C7/8 ORF minus the leader peptide was adapted for ligationinto pET-28b+ by designing adaptor-primers containing in-frame Nco I andXho I sites in the 5′- and 3′-primers, respectively. The complete 1337bp cDNA containing both the 5′- and 3′-UTRs was subjected to PCR (seeabove) with rTth DNA polymerase according to manufacturer's instructions(Perkin-Elmer) along with C7/8/pET-28b+/Forward(5′-CATGCATGCCATGGATCCGAGCATAACTGTGACACCTGATGAA-3′) (SEQ ID NO: 5) andC7/8/pET-28b+/Reverse(5′-GAGTCGCTCGAGATAAACTTTTAATAAGGCTGTGACTCTCCTTG-3′) (SEQ ID NO: 6)primers. After pET-adaption PCR was performed on the C7/8 cDNA fragmentand the resulting products separated on a 1.0% agarose gel, a 990 bpband was collected, restricted along with the pET-28b+vector with Nco Iand Xho I, reisolated on a agarose gel, ligated into the restrictedpET-28b+vector and transformed into Novablue (DE3) host cells.Recombinants were screened by direct ethidium bromide visualization(Data not shown) of restricted plasmid preparations and a clone waschosen for sequencing by the University of Virginia BiomolecularResearch Facility using primers surrounding the pET-28b+vector cloningsite to ascertain the proper insertion of the insert into thepET-28b+expression vector and the retention of the leaderless-C7/8reading frame.

[0119] Recombinant human C7/8 (rec-h-C7/8) containing a C-terminal(His)₆-Tag was isolated by resuspending the bacterial cell pellet in aBinding Buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris (pH 7.9)) priorto sonication and centrifugation. The pellet containing the insolubleprotein fraction and inclusion bodies was then resuspended in BindingBuffer made 6 M in urea, incubated on ice for 1 hr, resonicated andrecentrifuged prior to filtering the resulting supernatant through a0.45 m filter. The filtered bacterial protein lysate was then bound to a5 ml bed-volume His-Bind Resin (Novagen) column prepared according tomanufacturer's instructions and washed until the OD₂₈₀ approachedbaseline with Binding Buffer containing 6 M urea before washing thecolumn to baseline with Wash Buffer (40 mM imidazole, 0.5 M NaCl, 20 mMTris (pH 7.9) and 6 M urea). Final purification of the rec-h-C7/8 wasaccomplished by elution from the column with Elute Buffer (300 mMimidazole, 0.5 M NaCl, 20 mM Tris (pH 7.9)) and dialysis into 1×PBSprior to loading onto a Prep-Cell.

[0120] Large-scale preparation of purified recombinant human C7/8(rec-h-C7/8) was performed and the protein was prepared and solubilized.The rec-h-C7/8 was bound and batch-eluted from His Bind (Novagen) resinaccording to manufacturer's instructions. After column purification ofthe recombinant material the resulting eluted column fraction is greaterthan 99% pure. The partially purified rec-h-C7/8 was subjected toPrep-Cell electrophoresis resulting in the final product. The yield ofthe purified recombinant h-tekB1 was judged by SDS/PAGE electrophoresisto be approximately 1.7 mg of rec-h-tekB1/liter fermentation.

[0121] Generation of Rat Monospecific Polyclonal Sera

[0122] Nine young adult, female, virgin Lewis rats were immunized inthree groups with 100 μg of purified rec-h-C7/8 in either CompleteFreund s Adjuvant, alum or squalene monooleate. After 1 month each groupof 3 rats was boosted twice at 2 wk intervals with 50 μg of rec-h-C7/8in with the corresponding adjuvant and bled 1 wk after each boost.Specificity of the rat antisera for rec-h-C7/8 (α-rec-h-C7/8) was testedby Western blotting against rec-h-C7/8 as well as a SDS lystate of humansperm proteins separated on polyacrylamide gels and Western blotted.

EXAMPLE 2 Isolation of SAMP32

[0123] TritonX-114 Phase Partitioning and Two-Dimensional GelElectrophresis and Vectorial Labeling by Biotin

[0124] Human semen samples were obtained from healthy donor(s) asdescribed (Mandal et. al., 1999). Triton X-114 phase partitioning wasdone according to Bordier (Bordier, 1981). Two-dimensional gelelectrophresis was conducted as described in Example 1. Proteins werebiotin labeled using standard techniques.

[0125] SDS-PAGE and Western Blot

[0126] Proteins were separated first on discontinuous polyacrylamide gel(4% stacking and 15% separating gel) at 100 volts voltage constant on aBio-Rad mini-gel apparatus (Bio-Rad, California). Gels were then eitherstained with Coommassie blue or used for transfer to the membrane inwestern blot. For the western blot, proteins were transferred toNitrocellulose membrane (0.2 um) at 100 volts voltage constant for onehour with cooling. At completion, membranes were blocked in PBSTcontaining 5% non-fat milk, and 1% normal goat serum for one hour atroom temperature on a rocking platform. This procedure was followed byincubation of the membrane in PBST containing 5% non-fat milk, 1% normalgoat serum and 1:2,000 to 1:4,000 of primary antibody for another hour.After three washes of the membrane(s) with PBST, each for 10 min, themembranes were incubated further with PBST containing 5% non-fat milk 1%normal goat serum and 1:4,000 diluted secondary goat antibody conjugatedto HRP. Following three washes of the membrane(s) with PBST, the colorwas developed by adding TMB substrate at room temperature.

[0127] Screening of Lambda cDNA Library.

[0128] Lambda DR2 cDNA library from human testis was purchased fromClontech Inc. (Palo Alto, Calif.) and screened according to the usermanual. Briefly, a total of 2.5×10⁵ clones were plated out on six 150 mmagar plates on a bacteria lawn. After replica plate onto the nylonmembrane, phage particles were denatured in 0.5 M NaOH, 1.5M NaCl; andneutralized in 0.5M Tris pH 7.5, 1.5M NaCl. Following UV cross-link, thefilters were washed in 2×SSC, 0.2% SDS for 20 min at room temperature,prehybridized for 4 hours at 42° C. in hybridization solution, withoutthe probe, and hybridized at 42° C. overnight in the presence of 50%formamide. The 3′ 420 bp cDNA fragment, amplified and cloned from thehuman testis cDNA library, was used as a probe after labeling with³²P-dCTP by random primer method. Washes were done once in 2×SSC, 0.2%SDS for 20 min at RT, once in 0.2×SSC, 0.2% SDS for 20 min at 42° C.,and once in 0.2×SSC, 0.2% SDS for another 20 min at 50° C. At the end ofthe washes, the filters were exposed on X-ray film at −80° C. for 24 to48 hours before development. Secondary screening was repeated as aboveto isolate single, pure phage plaques. Finally, the cDNA inserts wererecovered as plasmids in pDR2 vector from the phage particles bytransforming the E. coli AM1 strain expressing Cre recombinase. Thisprocess was made possible by the presence of two phage P1 derived lox-Precombination sites at both ends of the cDNA insert.

[0129] Polymerase Chain Reaction (PCR) and Rapid Amplification of cDNAEnds(RACE)

[0130] Polymerase chain reactions were done following standardconditions (Hao et. al., 1997). Hot start PCRs' using the librarytemplate and for RACE were performed by using the Amp-Taq DNA polymerasefrom Perkin-Elmer. The parameters used were 94° C. 10 min, 94° C. 30sec, 55° C. 30 sec, and 72° C., 2-4 min for 35-40 cycles. PCRs usingplasmid template were done using cloned pfu DNA polymerse fromStratagene (parameters were the same except the denaturation was 5 minbefore cycling and the cycle number was 30 cycles). The primers used toamplify the probe for plaque and northern blot hybridization were Hao 1(AGTCACCCCTTGGCTTTCGAGT; SEQ ID NO: 10) and Hao2(AATATTCTGTAATATCCTTTGGTT; SEQ ID NO: 11). The primers for SAMP32 3′amplification of were Hao39 (CTTTGTATGTCACATTCCCTGAAG; SEQ ID NO: 12)and Hao41 (GAGGTACAATCCGAGCAGAGTTCT; SEQ ID NO: 13) or Hao41 and APIprimer supplied in the Marathon ready cDNA kit (Clontech).

[0131] Northern and Dot Blot

[0132] Human multiple tissue northern membrane containing 8 tissues andMulti-tissue array RNA dots containing 67 tissues were purchased fromClontech Inc. The same 3′ 420 bp cDNA described above was used as probe.Hybridization was performed in ExpressHyb™ solution at 68° C. for 1 hourand washed successively two times in 2×SSC, 0.1% SDS each for 20 min atroom temperature; and three times in 0.1×SSC, 0.1% SDS each for 20 minat 65° C. Films were exposed 24 to 72 hours at −80° C. The same membranewas probed with beta-actin supplied in the kit after stripping out theprevious probe.

[0133] To probe the multi-tissue array RNA dots, the same 420 bp of DNAwas labeled and hybridization was done at 68° C. overnight in ExpressHybsolution containing salmon sperm ssDNA and Cot-1 DNA overnight,following manufacturer's instruction. The film was exposed 96 hours at−80° C.

[0134] Expressing Recombinant Protein in E. coli and Production ofAntiserum

[0135] To express SAMP32 in E. coli, the fragment that contains aminoacid 30-221 of the open reading frame was amplified by PCR. It was thenfused in frame with the His-tag at both ends of pET28b using the NheIand XhoI sites. Construct was verified by DNA sequencing. Due to thecodon bias, Epicurian Coli BL21-CodonPlus™ cells were used as the hoststrain (Stratagene) in the place of conventional BL21 DE3. The plasmidwas transformed into the host strain and a large culture derived from asingle colony, was grown to OD600=1.0 at 37° C. in LB in the presence of50 ug/ml of Kanamycin. Isopropyl-1-thio-beta-D-Glactopyranoside (ITPG)was then added to a final concentration of 1 mM to induce expression.After a further 3 to 4 hours of growth, the bacteria were collected bycentrifugation for recombinant protein processing. The recombinantprotein was affinity purified with Ni-NTA column in denatured condition.Preparative gel electrophresis couple with Elutip™ was employed toremove minor contaminant proteins from E. Coli.

[0136] Female virgin Lewis rats, weighing 160-200 grams, were used inantiserum production. Purified recombinant protein in PBS was emulsifiedwith equal amount(s) of Freud's complete adjuvant and each rat wasinjected with 100 ug of protein in 0.3 ml s.c. initially. The sameamounts of protein, emulsified with equal volume of Freud's incompleteadjuvant, were given by the same route in subsequent booster injectionsat two weeks interval. The antibody titers were checked by western blotanalysis 10 days after each booster injection. The animals weresacrificed soon after a check indicated a titer above 1:2000 on totalsperm extract western.

[0137] Immunofluorescence of Enzyme Dissociated Tissue

[0138] Human testis block was cut and dissociated with collagenase,micrococcal nuclease and trypsin. Dissociated cells were spread out onslides and dried. The slides were washed in PBS trice, permeablized inMethanol, and washed again with PBS. The slides were blocked in 10%normal donkey serum for 1 hour. They were then incubated with antibodysolution containing 1% normal donkey serum and 1:100 to 1:200 dilutionof rat anti-SAMP32 antiserum overnight at 4° C. After three washes withPBST containing 1% normal donkey serum, the slides were incubated withsecondary antibody (donkey anti-rat conjugated with TRITC, 1:200 in PBSTand 1% normal donkey serum) for 60 min. The slides were washed twicewith PBST, twice with PBS, and fixed with 2% paraformaldehyde for 10min. After washing with PBS, they were incubated in equilibrationbuffer. Nucleus were stained with DAPI and slides were mounted withSlow-Fade anti-fade reagent (Molecular Probes). Images were capturedusing a Zeiss Axioplan2 microscope equipped with epifluorescence usingOpen Lab software (Improvision).

[0139] Preparation of Spermatozoa and Acrosomal Reaction

[0140] Human semen samples were obtained from healthy donors and Swim-upsperm was prepared as follows. Freshly ejaculated semen samples wereliquidified for 30 min to 1 hour at room temperature. They were dilutedwith an equal volume of BWW and centrifuged at 500 g for 5 min to removeseminal plasma. The sperm pellets were washed twice with BWW containing10 mg/ml of BSA (500 g×5 min). Following the removal of supernatant, thesperm pellets were overlaid carefully with BWW containing 30 mg/ml ofhuman serum albumin. The sperm samples were then incubated at 37° C. for1 hour to allow the sperm to swim up. Sperm were then incubated in BWWin the presence of calcimycin to induce acrsomal reaction at 37° C.

[0141] Immunofluorescence Localization of SAMP32 in Sperm and ElectronMicroscopic Analysis

[0142] The sperm were air-dried onto Poly-L-Lysine coated slides(Polysciences, Warrington, Pa.). They were permeabilized in methanol,air-dried and blocked in 10% normal goat serum in PBST for 30 min. Thesperm were then incubated with 1:100 dilution of anti-SAMP32 primaryantiserum for 2 hours at 37° C. Following incubation with the secondarygoat-anti-Rat IgG conjugated with Cy3 at 1:100 dilution for 1 hour at37° C., the slides were washed in PBS, coated with slow-fade, andmounted in coverslips.

[0143] For immuno-electron microscopy, pooled sperm were washed twice inHam's F-10. containing 3% sucrose. They were then fixed in 4%paraformaldehyde and 0.2% glutaraldehyde in wash buffer for 15 min at22° C. The fixatives were removed by washing and the sperm weredehydrated by passing through a series of graded ethanol, ranging from40% to 100%. Following embedding in Lowicryl K4M, the blocks werepolymerized with UV light at −20° C. for 72 hours and ultra-thinsections were cut. To stain the ultra-thin sections, they were firstblocked in undiluted normal goat serum for 15 min at 22° C. They werethen incubated for 16 hours at 4° C. with either preimmune orrat-anti-SAMP32 antiserum at the dilution of 1:50 containing 1% normalgoat serum, 1% BSA and 0.1% Tween-20. After washing, the sections werethen incubated with 1:100 dilution of 5 nm gold-conjugated goat-anti IgG(Goldmark Biologicals, Phillipsburg, N.J.) for 1.5 hours at 22° C. Thesections were washed in distilled water and stained with uranyl acetateand observed with the JOEL 100CX electron microscope.

EXAMPLE 3 Isolation of C58

[0144] Separation of Hydrophobic, Putative Sperm Membrane AssociatedProteins by TX-114-Phase Partitioning

[0145] To isolate the hydrophobic, putative membrane associated spermproteins, TX-114-phase partitioning was conducted. In particular, humansperm cells were solubilized in 1.7% TX-114/TBS at 4° C. The solutionwas cetrifuged and the supernatant containing the solubilized proteinswas recovered. TBS was added to adjust the TX-114 concentration to 1.%.Warming to 30° C. allowed the aggregation of micelles and separation ofdetergent phase from the aqueous phase. The two phases were separated bycentrifugation.

[0146] 2-D electrophoresis was used to analyze the phase partitionedsperm proteins. The starting total extract, aqueous phase extract, anddetergent phase extract were analyzed by 2-D gel electrophoresis. TX-114phase partitioning allowed the selective partitioning of severalhydrophobic proteins to the detergent phase. C58 is one of the 2-Dprotein spots enriched in the hydrophobic detergent phase extract.

[0147] To determine if C58 is a surface protein, freshly harvested humansperm cells were vectorially labeled with sulfo-NHS-LC biotin, subjectedto TX-114 phase partitioning and the detergent phase extract obtainedwas separated by 2-D gel electrophoresis. Protein spots labeled withbiotin were visualized by avidin-ECL and compared to a silver stainedcompanion gel to identify the biotinylated protein spots. The 2D gelsindicated that C58 is vectorially labeled with sulfo-NHS-LC biotin, andthus C58 is probably located on the surface of the sperm.

[0148] The C58 spot excised from a Coomassie stained 2-D gel wasmicrosequenced using tandem mass spectrometry. Four peptide sequenceswere isolated. Data base search analysis done using the tryptic peptidesrevealed no matches to any known proteins. However, two of the peptidesmatched to a Human testis EST clone (AC. No. AA778671; SEQ ID NO: 17).

[0149] Cloning of C58 cDNA Utilizing EST cDNA Sequence

[0150] Human testis EST (AC NO. AA778671) matching to tryptic peptidesobtained from microsequeincing of spot C58 was amplified by PCR. The PCRcDNA fragment was verified and then labeled with ³²P and used to screena λ-DR2-testis cDNA library. Positive clones were isolated andsequenced. The cDNA for the complete open reading frame of C58 wasobtained (SEQ ID NO: 14). The Complete ORF of C58 contained 372 basepairs encoding 124 amino acids with a predicted Mol Wt. of 13 and apredicted pI of 5.5. Sequences of one of the tryptic peptidesoriginating from the cored 2-D spot was found embedded in the ORF(ATSCGLEEPVSYR; SEQ ID NO: 19). Alignment of amino acid sequence of C58with other proteins having a similar domain utilizing a bioinformaticprogram ‘Multialign’ revealed several conserved cysteine residues werefound among the group of proteins showing similarity to C58. Theconserved position of the cysteine residues, which may be involved inthe formation of disulfide bridges, suggests a potential conservedfunction secondary and tertiary structure to these proteins. Internalsequence of all proteins showed similarity to the Ly-6/UPAR superfamilyof proteins.

[0151] C58 Shows homology to Ly6D (E48 Antigen)_Human (see FIG. 3). Ly6Dis expressed exclusively at the outer cell surface of transitionalepithelial and the keratinocytes of stratified squamous epithelialcarcinoma. The protein is attached to the cellular membrane by aGPI-anchor. The Ly6D protein contains a 1UPAR/Ly-6 Domain and isbelieved to be involved in cell-cell adhesion and signal transduction.

[0152] A bioinformatic analysis of amino acid sequence of C58 was doneto check the possibility of it being a Glycosyl Phosphatidyl Inositolanchored protein. The structure of C58 indicates that the protein is aGPI anchored Protein. In particular, C58 has a transmembrane N-terminalsignal peptide (amino acids 1-19 of SEQ ID NO: 16), a transmembraneC-terminal hydrophobic domain and a transamidase cleavage site (locatedat amino acids 97-99 of SEQ ID NO: 16) near the C-terminal end typicalof a GPI anchored protein. Amino acids 22-112 of C58 (SEQ ID NO: 16)include a Ly-6 Antigen/Urokinase Plasminogen Activator receptor-likedomain.

[0153] Northern Blot Analysis for Tissue Specificity

[0154] A standard Northern blot analysis was done by using Clontech'smultiple tissue northern blot analysis panel of 8 human tissues (seeFIG. 4). cDNA from the entire open reading frame of C58 was used as theprobe (radiolabeled with P³²) and hybridized to 2 ug poly-(A)+ mRNAsisolated from spleen, thymus, prostate, testis, ovary, small intestinemcolon and leucocyte and located in lanes 1-8, respectively. Size ofmolecular weight markers is indicated at left. A positive signal wasobtained in the testis lane only for standard Northern blot analysis. AMTE array Northern blot analysis of 76 tissues was done also using cDNAfrom entire ORF of C58 as the probe. Similarly a 76 dot-blot Northernblot analysis also yielded a prominent signal in the testis box only.Thus the message for C58 is transcribed in testis only.

[0155] Recombinant Expression of C58

[0156] Two different constructs were made for the recombinant expressionof C58 using vector pET 28b. Construct 1 (C58-1): cDNA corresponding toaa 20-124 was engineered into the vector with a histidine tag at theC-terminal end. Construct 2 (C58-2): cDNA corresponding to aa 20-100 wasengineered into the vector with a histidine tag at the C-terminal end.The constructs were trasfected into E. coli and a small scale culture ofthe host cells BL21-pLys S-DE3 containing vector pET 28b with or withoutinsert (Control) were induced with 1 mM IPTG and harvested after 4hours. Bacterial lysates made in Laemmli buffer were run on 16%SDS-PAGE. The electrophoresed proteins were Coomassie stained ortransferred to a nitrocellulose membrane and probed with Ni-NTA (1:2K)to visualize expressed protein.

[0157] Upon induction, additional bands of proteins were visualized on aCoomassie stained gel on the induced lanes corresponding to C58-1 andC58-2, which also reacted with Ni-NTA on a western blot. The bandsappeared at the expected molecular weight range of 9.5-14 kDa.

[0158] Four-liter cultures of host cells expressing C58-1 and C58-2 weremade and induced with 1 mM IPTG. The expressed proteins were partiallypurified by His-column chromatography. The proteins were furtherpurified by electrophoretic purification using Prep Cell (BioRad).Samples from all the fractions were analyzed by SDS-PAGE. His-Columnchromatography followed by Prep Cell purification allowed thepurifaction of expressed protein.

[0159] Western Blotting Analysis of Recombinant C58 and Human SpermProteins with Anti-Rat Antibody for Recombinant C58.

[0160] Human sperm proteins were solubilized in Celis extraction buffer.The supernatant was mixed with equal volume of 2× Laemmli buffer.Samples of recombinant proteins were prepared by directly solubilizingthe lyophilyzed powder of recombinant C58 (+transmembrane domain; (TM))and C58 (−TM) in 1× Laemmli buffer. Samples were run on 16% SDS-PAGE.The proteins transferred to a nitrocellulose membrane were probed withpreimmune (PI) and immune (IM) serum from rat #2 for C58 (+TM) and rat#17 for C58 (−TM). Blots were incubated with secondary antibody (goatanti rat IgG) and developed by using TMB membrane peroxidase substrate.Strips were also incubated directly with secondary antibody (sec. Abcon) to visualize any non-specific proteins reacting with secondaryantibody alone.

[0161] Antibodies for both C58 (+TM) and C58 (−TM) recognized therespective recombinant proteins. Both antibodies recognized a singleband at approximately 12.5 kDa on the sperm protein blots indicatingthat the antibodies are recognizing the native C58 protein.

[0162] Western Blot Analysis of Human Sperm Proteins Separated by 2-DGel Electrophoresis Using Anti-Rat Recombinant C58 Antibody.

[0163] Human sperm proteins were solubilized with Celis extractionbuffer, separated by 2-D gel electrophoresis and transferred tonitrocellulose membrane. The proteins were stained with protogold tovisualize total separated proteins. The blots were probed with anti-ratantibody for recombinant C58 (−TM) (rat #17) at 1:4K. The blots wereincubated with secondary antibody and the reactive spots were visualizedusing TMB membrane peroxidase substrate.

[0164] The antibody reacted with the native C58 spot that was initiallycored for microsequencing. In addition to recognizing the cored proteinit also recognized 2 small additional spots on either side at the samemolecular weight range suggesting the possibility of the protein havingcharge variants

[0165] Indirect Immunofluorescence Localization of C58 on Air-driedHuman Sperm Using Anti-Rat Recombinant C58 (+TM) Antibody.

[0166] Human sperm were prepared by swim-up method, and air-dried onslides at a concentration of ˜2×10⁶. The sperm were blocked with 10%normal goat serum in PBS and incubated with anti-rat antibody (rat #2)for C58 (+TM) at 1:30 dilution. The slides were washed in PBS andincubated with FITC conjugated goat anti-rat IgG. Then they were washedin PBS and mounted using antifade.

[0167] Sperm incubated with immune serum showed signal over the entiresurface of the sperm with higher reactivity localized to the head. Therewas no fluorescent signal observed with the sperm smear incubated withpreimmune serum. The results indicated that C58 is located on thesurface of the sperm

EXAMPLE 4 Human Sperm Binding and Fusion Assay Using Zona-Free HamsterEggs

[0168] Sperm Preparation:

[0169] Motile sperm were harvested by the swim up method of Bronson andFusi (1990). Briefly, a 500 ml sperm sample underlaid in 2 ml of BWWmedia containing 5 mg/ml HSA. Sperm were allowed to swim up for 1.5-2 h.Swimup sperm were collected and 8 ml of BWW+5 mg/ml HSA was added. Thecomposition was spin at 600×g for 8 min at RT, the supernatant wasremoved and 8 ml of media was added to the pellet. The resuspendedpellet was spun at 600×g for 8 min at RT. The supernatant was removedand 50 ml of BWW containing 30 mg/ml HSA was added to the pellet. Totalsperm cells were counted and then incubated overnight in BWW+30 mg/mlHSA at a concentration of 20×10⁶ sperm/ml.

[0170] Egg Collection:

[0171] Female hamsters received i.p. injections of 30 IU PMSG followedby 30 IU of hCG 72 h later. 14-16 h following hCG injection, hamsterswere sacrificed and oviducts are collected in BWW media containing 5mg/ml HSA. Cumulus cells were removed with 1 mg/ml hyaluronidase, theeggs were washed and zona pellucidae removed with 1 mg/ml trypsin. Theeggs were then thoroughly washed and allowed to rest in the incubator.

[0172] Sperm/Antibody Incubation:

[0173] Sperm was diluted to 20×10⁶ sperm/ml and incubated withappropriate dilutions of pre-immune or immune sera (initially a 1:10 and1:50 dilution of sera is tested) in paraffin oil covered microdrops for1 h.

[0174] Hamster eggs were added to the drops containing thesperm+antibody. The gametes were then co-incubated for 3 h.

[0175] Assessment of Binding and Fusion:

[0176] Eggs were washed free of unbound and loosely bound sperm byserial passage through 5 (50 ml) wash drops. The same pipet is used forall eggs washed in an individual experiment. Eggs are then stained byshort-term (5-15 s) exposure to 1 mM acridine orange-3% DMSO in BSA/BWW(30 mg/ml), washed through 4 (50 ml) wash drops and mounted under 22×22mm coverslips. Under UV illumination, unexpanded head s ofoolemma-adherant sperm were counted and sperm that had penetrated theooplasm exhibited expanded green heads. All experiments were repeated 3times

1:10 Dilution of C 71 Antibody Number of Sperm Bound Per Egg

[0177] Pre Immune 24.2 Immune 11.5

[0178] P value=6.6×10⁵

Number of Sperm Fused Per Egg

[0179] Pre Immune 2.2 Immune 0.35

[0180] P value=1.8×10⁶

1:10 Dilution of C7/8 Antibody Number of Sperm Bound Per Egg

[0181] Pre Immune 13.7 Immune 7.9

[0182] P value=0.003

Number of Sperm Fused Per Egg

[0183] Pre Immune 1.8 Immune 0.59

[0184] P value=9.63×10⁵

1 20 1 1337 DNA Homo sapiens 1 ccagcctggt ggccccagga cgttccggtcgcatggcaga atgctggggg cgacgcctat 60 gaagccctta gtccttctag ttgcgcttttgctatggcct tcgtctgtgc cggcttatcc 120 gagcataact gtgacacctg atgaagagcaaaacttgaat cattatatac aagttttaga 180 gaacctagta cgaagtgttc cctctggggagccaggtcgt gagaaaaaat ctaactctcc 240 aaaacatgtt tattctatag catcaaagggatcaaaattt aaggagctag ttacacatgg 300 agacgcttca actgagaatg atgttttaaccaatcctatc agtgaagaaa ctacaacttt 360 ccctacagga ggcttcacac cggaaataggaaagaaaaaa cacacggaaa gtaccccatt 420 ctggtcgatc aaaccaaaca atgtttccattgttttgcat gcagaggaac cttatattga 480 aaatgaagag ccagagccag agccggagccagctgcaaaa caaactgagg caccaagaat 540 gttgccagtt gttactgaat catctacaagtccatatgtt acctcataca agtcacctgt 600 caccacttta gataagagca ctggcattgagatctataca gaatcagaag atgttcctca 660 gctctcaggt gaaactgcga tagaaaaacccgaagagttt ggaaagcacc cagagagttg 720 gaataatgat gacattttga aaaaaattttagatattaat tcacaagtgc aacaggcact 780 tcttagtgac accagcaacc cagcatatagagaagatatt gaagcctcta aagatcacct 840 aaaacccagc cttgctctag cagcagcagcagaacataaa ttaaaaacaa tgtataagtc 900 ccagttattg ccagtaggac gaacaagtaataaaattgat gacatcgtaa ctgttattaa 960 catgctgtgt aattctagat ctaaactctatgaatattta gatattaaat gtgttccacc 1020 agagatgaga gaaaaagctg ctacagtattcaatacatta aaaaatatgt gtagatcaag 1080 gagagtcaca gccttattaa aagtttattaaacaataata taaaaatttt aaacctactt 1140 gatattccat aacaaagctg atttaagcaaactgcatttt ttcacaggag aaataatcat 1200 attcgtaatt tcaaaagttg tataaaaatattttctattg tagttcaaat gtgccaacat 1260 ctttatgtgt catgtgttat gaacaattttcatatgcact aaaaacctaa tttaaaataa 1320 aattttggtt caggaaa 1337 2 350 PRTHomo sapiens 2 Met Lys Pro Leu Val Leu Leu Val Ala Leu Leu Leu Trp ProSer Ser 1 5 10 15 Val Pro Ala Tyr Pro Ser Ile Thr Val Thr Pro Asp GluGlu Gln Asn 20 25 30 Leu Asn His Tyr Ile Gln Val Leu Glu Asn Leu Val ArgSer Val Pro 35 40 45 Ser Gly Glu Pro Gly Arg Glu Lys Lys Ser Asn Ser ProLys His Val 50 55 60 Tyr Ser Ile Ala Ser Lys Gly Ser Lys Phe Lys Glu LeuVal Thr His 65 70 75 80 Gly Asp Ala Ser Thr Glu Asn Asp Val Leu Thr AsnPro Ile Ser Glu 85 90 95 Glu Thr Thr Thr Phe Pro Thr Gly Gly Phe Thr ProGlu Ile Gly Lys 100 105 110 Lys Lys His Thr Glu Ser Thr Pro Phe Trp SerIle Lys Pro Asn Asn 115 120 125 Val Ser Ile Val Leu His Ala Glu Glu ProTyr Ile Glu Asn Glu Glu 130 135 140 Pro Glu Pro Glu Pro Glu Pro Ala AlaLys Gln Thr Glu Ala Pro Arg 145 150 155 160 Met Leu Pro Val Val Thr GluSer Ser Thr Ser Pro Tyr Val Thr Ser 165 170 175 Tyr Lys Ser Pro Val ThrThr Leu Asp Lys Ser Thr Gly Ile Glu Ile 180 185 190 Tyr Thr Glu Ser GluAsp Val Pro Gln Leu Ser Gly Glu Thr Ala Ile 195 200 205 Glu Lys Pro GluGlu Phe Gly Lys His Pro Glu Ser Trp Asn Asn Asp 210 215 220 Asp Ile LeuLys Lys Ile Leu Asp Ile Asn Ser Gln Val Gln Gln Ala 225 230 235 240 LeuLeu Ser Asp Thr Ser Asn Pro Ala Tyr Arg Glu Asp Ile Glu Ala 245 250 255Ser Lys Asp His Leu Lys Pro Ser Leu Ala Leu Ala Ala Ala Ala Glu 260 265270 His Lys Leu Lys Thr Met Tyr Lys Ser Gln Leu Leu Pro Val Gly Arg 275280 285 Thr Ser Asn Lys Ile Asp Asp Ile Val Thr Val Ile Asn Met Leu Cys290 295 300 Asn Ser Arg Ser Lys Leu Tyr Glu Tyr Leu Asp Ile Lys Cys ValPro 305 310 315 320 Pro Glu Met Arg Glu Lys Ala Ala Thr Val Phe Asn ThrLeu Lys Asn 325 330 335 Met Cys Arg Ser Arg Arg Val Thr Ala Leu Leu LysVal Tyr 340 345 350 3 22 DNA Artificial Sequence Description ofArtificial Sequence PCR primer 3 cttgctctag cagcagcaga ac 22 4 30 DNAArtificial Sequence Description of Artificial Sequence PCR Primer 4tcataacaca tgacacataa agatgttggc 30 5 43 DNA Artificial SequenceDescription of Artificial Sequence PCR Primer 5 catgcatgcc atggatccgagcataactgt gacacctgat gaa 43 6 44 DNA Artificial Sequence Description ofArtificial Sequence PCR Primer 6 gagtcgctcg agataaactt ttaataaggctgtgactctc cttg 44 7 14 PRT Homo sapiens 7 Ala Ser Thr Pro Glu Val GlnSer Glu Gln Ser Ser Val Arg 1 5 10 8 1455 DNA Homo sapiens 8 gcgcttcgacgtacctgtcc tcaggagccg cggcggcgac tgcgcctcgg acggccgtcg 60 gggccgagaaccatgagccc caggggcacg ggctgctccg ccgggctgct gatgactgtc 120 ggctggctgcttctggcggg cctccagtcc gcgcgcggga ccaacgtcac cgctgccgtc 180 caggatgccggcctggccca cgaaggcgag ggcgaggagg agaccgaaaa caacgacagc 240 gagaccgcggagaactacgc tccgcctgaa accgaggatg tttcaaatag gaatgtcgtc 300 aaagaagtagaattcggaat gtgcaccgtt acatgtggta ttggggttag agaagttata 360 ttaacaaatggatgccctgg tggtgaatcc aagtgtgttg tacgggtaga agaatgccgt 420 ggaccaacagattgtggctg gggtaaacca atttcagaaa gtcttgaaag tgttagattg 480 gcatgtattcacacatctcc cttaaatcgt ttcaaatata tgtggaaact tctaagacaa 540 gaccaacaatccattatact tgtaaatgat tcagcaatcc tagaagtacg caaggaaagt 600 caccccttggctttcgagtg tgacacactg gataataatg aaatagtagc aactattaaa 660 ttcacagtctatacgagcag tgaattgcag atgagaagat caagcctacc agccactgat 720 gcagccctaatttttgtgct gaccatagga gtcattatct gtgtatttat aattttctta 780 ttgatcttcataatcataaa ttgggcagca gtcaaggctt tttggggggc aaaagcctct 840 acacctgaggtacaatccga gcagagttct gtgagataca aagattcaac ttctcttgac 900 caattaccaacagaaatgcc tggtgaagat gatgctttaa gtgaatggaa tgaatgatgt 960 ttgaatgatatataacaaac caaaggatat tacagaatat tagattcatt attacaaaaa 1020 taaaatacacattgaaatac tttaataatg ttgcgatgga ttgccacagt gtgaaggaaa 1080 tgcagtgtggggataggact attttatcag tgcatttttc cagtacagtt atcaaatatt 1140 acttttaatttgttctcaac acttatttca ggtaatagct tggggatatt tatctaaagt 1200 acccccaacaaatcttctaa gtgcattttt gatcactttg ataacttctt aggtgatttg 1260 cctgttttgtcttaaataag aacaatgtaa tatagaaatg ctttacatat tagactttct 1320 ctcccctggaagcactgggt tgaacttgct aaagtaaatc atactttaga atctcttcag 1380 ggaatgtgacatacaaagtt tgtaagacat gaagtaataa cgataatgat aacaataaat 1440 gcttacttagtgaaa 1455 9 294 PRT Homo sapiens 9 Met Ser Pro Arg Gly Thr Gly Cys SerAla Gly Leu Leu Met Thr Val 1 5 10 15 Gly Trp Leu Leu Leu Ala Gly LeuGln Ser Ala Arg Gly Thr Asn Val 20 25 30 Thr Ala Ala Val Gln Asp Ala GlyLeu Ala His Glu Gly Glu Gly Glu 35 40 45 Glu Glu Thr Glu Asn Asn Asp SerGlu Thr Ala Glu Asn Tyr Ala Pro 50 55 60 Pro Glu Thr Glu Asp Val Ser AsnArg Asn Val Val Lys Glu Val Glu 65 70 75 80 Phe Gly Met Cys Thr Val ThrCys Gly Ile Gly Val Arg Glu Val Ile 85 90 95 Leu Thr Asn Gly Cys Pro GlyGly Glu Ser Lys Cys Val Val Arg Val 100 105 110 Glu Glu Cys Arg Gly ProThr Asp Cys Gly Trp Gly Lys Pro Ile Ser 115 120 125 Glu Ser Leu Glu SerVal Arg Leu Ala Cys Ile His Thr Ser Pro Leu 130 135 140 Asn Arg Phe LysTyr Met Trp Lys Leu Leu Arg Gln Asp Gln Gln Ser 145 150 155 160 Ile IleLeu Val Asn Asp Ser Ala Ile Leu Glu Val Arg Lys Glu Ser 165 170 175 HisPro Leu Ala Phe Glu Cys Asp Thr Leu Asp Asn Asn Glu Ile Val 180 185 190Ala Thr Ile Lys Phe Thr Val Tyr Thr Ser Ser Glu Leu Gln Met Arg 195 200205 Arg Ser Ser Leu Pro Ala Thr Asp Ala Ala Leu Ile Phe Val Leu Thr 210215 220 Ile Gly Val Ile Ile Cys Val Phe Ile Ile Phe Leu Leu Ile Phe Ile225 230 235 240 Ile Ile Asn Trp Ala Ala Val Lys Ala Phe Trp Gly Ala LysAla Ser 245 250 255 Thr Pro Glu Val Gln Ser Glu Gln Ser Ser Val Arg TyrLys Asp Ser 260 265 270 Thr Ser Leu Asp Gln Leu Pro Thr Glu Met Pro GlyGlu Asp Asp Ala 275 280 285 Leu Ser Glu Trp Asn Glu 290 10 22 DNAArtificial Sequence Description of Artificial Sequence PCR Primer 10agtcacccct tggctttcga gt 22 11 24 DNA Artificial Sequence Description ofArtificial Sequence PCR Primer 11 aatattctgt aatatccttt ggtt 24 12 24DNA Artificial Sequence Description of Artificial Sequence PCR Primer 12ctttgtatgt cacattccct gaag 24 13 24 DNA Artificial Sequence Descriptionof Artificial Sequence PCR Primer 13 gaggtacaat ccgagcagag ttct 24 14600 DNA Homo sapiens 14 gtcccggatc cgcgagggac gcagggcgtt gggaacagaggacactccag gcgctgaccc 60 tgggaggcca ggaccagggc caaagtcccg tgggcaagaggagtcctcag aggtccttca 120 ttcagcggtt ccgggaggtc tgggaagccc acggcctggctggggcaggg tcaacgccgc 180 caggccgcca tggtcctgtg ctggctgctg cttctggtgatggctctgcc cccaggcacg 240 acgggcgtca aggactgcgt cttctgtgag ctcaccgactccatgcagtg tcctggtacc 300 tacatgcact gtggcgatga cgaggactgc ttcacaggccacggggtcgc cccgggcact 360 ggtccggtca tcaacaaagg ctgcctgcga gccaccagctgcggccttga ggaacccgtc 420 agctacaggg gcgtcaccta cagcctcacc accaactgctgcaccggccg cctgtgtaac 480 agagccccga gcagccagac agtgggggcc accaccagcctggcactggg gctgggtatg 540 ctgcttcctc cacgtttgct gtgaccaaca gggaggacagggcctgggac tgttcttcca 600 15 375 DNA Homo sapiens 15 atggtcctgtgctggctgct gcttctggtg atggctctgc ccccaggcac gacgggcgtc 60 aaggactgcgtcttctgtga gctcaccgac tccatgcagt gtcctggtac ctacatgcac 120 tgtggcgatgacgaggactg cttcacaggc cacggggtcg ccccgggcac tggtccggtc 180 atcaacaaaggctgcctgcg agccaccagc tgcggccttg aggaacccgt cagctacagg 240 ggcgtcacctacagcctcac caccaactgc tgcaccggcc gcctgtgtaa cagagccccg 300 agcagccagacagtgggggc caccaccagc ctggcactgg ggctgggtat gctgcttcct 360 ccacgtttgctgtga 375 16 124 PRT Homo sapiens 16 Met Val Leu Cys Trp Leu Leu Leu LeuVal Met Ala Leu Pro Pro Gly 1 5 10 15 Thr Thr Gly Val Lys Asp Cys ValPhe Cys Glu Leu Thr Asp Ser Met 20 25 30 Gln Cys Pro Gly Thr Tyr Met HisCys Gly Asp Asp Glu Asp Cys Phe 35 40 45 Thr Gly His Gly Val Ala Pro GlyThr Gly Pro Val Ile Asn Lys Gly 50 55 60 Cys Leu Arg Ala Thr Ser Cys GlyLeu Glu Glu Pro Val Ser Tyr Arg 65 70 75 80 Gly Val Thr Tyr Ser Leu ThrThr Asn Cys Cys Thr Gly Arg Leu Cys 85 90 95 Asn Arg Ala Pro Ser Ser GlnThr Val Gly Ala Thr Thr Ser Leu Ala 100 105 110 Leu Gly Leu Gly Met LeuLeu Pro Pro Arg Leu Leu 115 120 17 569 DNA Homo sapiens 17 gcactggtccggtcatcaac aaaggctgcc tgcgagccac cagctgcggc cttgaggaac 60 ccgtcagctacaggggcgtc acctacagcc tcaccaccaa ctgctgcacc ggccgcctgt 120 gtaacagagccccgagcagc cagacagtgg gggccaccac cagcctggca ctggggctgg 180 gtatgctgcttcctccacgt ttgctgtgac caacagggag gacagggcct gggactgttc 240 tcccagatccgccactcccc atgtccccat gtccttcccc cactaaatgg ccagagaggc 300 cctggacaacctcttgcggc cctggcttca tcccttctaa ggctgtccac caggagcccg 360 gtgctaggggaagcatcccc aggcctgact gagcggcagg ggagcacggc ccgtgggttt 420 gattgtattactctgttcca ctggttctaa gacgcagagc ttctcacatc tcaatcagga 480 tgcttctctccattggtagc actttagagt ccatgaaata tggtaaaaaa tatatatata 540 18 166 DNAHomo sapiens 18 atggtcctgt gctggctgct gcttctggtg atggctctgc ccccaggcacgacgggcgtc 60 aaggactgcg tcttctgtga gctcaccgac tccatgcagt gtcctggtacctacatgcac 120 tgtggcgatg acgaggactg cttcacaggc cacggggtcg ccccgg 166 1913 PRT Homo sapiens 19 Ala Thr Ser Cys Gly Leu Glu Glu Pro Val Ser TyrArg 1 5 10 20 128 PRT Homo sapiens 20 Met Arg Thr Ala Leu Leu Leu LeuAla Ala Leu Ala Val Ala Thr Gly 1 5 10 15 Pro Ala Leu Thr Leu Arg CysHis Val Cys Thr Ser Ser Ser Asn Cys 20 25 30 Lys His Ser Val Val Cys ProAla Ser Ser Arg Phe Cys Lys Thr Thr 35 40 45 Asn Thr Val Glu Pro Leu ArgGly Asn Leu Val Lys Lys Asp Cys Ala 50 55 60 Glu Ser Cys Thr Pro Ser TyrThr Leu Gln Gly Gln Val Ser Ser Gly 65 70 75 80 Thr Ser Ser Thr Gln CysCys Gln Glu Asp Leu Cys Asn Glu Lys Leu 85 90 95 His Asn Ala Ala Pro ThrArg Thr Ala Leu Ala His Ser Ala Leu Ser 100 105 110 Leu Gly Leu Ala LeuSer Leu Leu Ala Val Ile Leu Ala Pro Ser Leu 115 120 125

1. A purified polypeptide comprising the amino acid sequence of SEQ IDNO: 2; an amino acid sequence that differs from SEQ ID NO: 2 by one ormore conservative amino acid substitutions; or an amino acid sequencethat differs from SEQ ID NO: 2 by a single mutation, wherein the singlemutation represents a single amino acid deletion, insertion orsubstitution.
 2. A purified or recombinant polypeptide comprising anamino acid sequence of SEQ ID NO:
 2. 3. A nucleic acid sequencecomprising the sequence of SEQ ID NO:
 1. 4. A transgenic host cellcomprising the nucleotide sequence of claim
 3. 5. A nucleic acidsequence comprising a 100 bp nucleic acid sequence that is identical toa contiguous 100 bp sequence of SEQ ID NO:
 1. 6. A method of screeningfor potential human therapeutic agents, said method comprisingcontacting a C7/8 polypeptide with a candidate compound; and determiningif the candidate compound selectively binds to the C7/8 polypeptide. 7.The method of claim 6 wherein the C7/8 polypeptide is expressed on thesurface of a cell.
 8. An antibody that binds specifically to the proteinof SEQ ID NO:
 2. 9. An antigenic composition comprising a C7/8polypeptide and a pharmaceutically acceptable carrier.
 10. A purifiedpolypeptide comprising the amino acid sequence of SEQ ID NO: 9; an aminoacid sequence that differs from SEQ ID NO: 9 by one or more conservativeamino acid substitutions; or an amino acid sequence that differs fromSEQ ID NO: 9 by a single mutation, wherein the single mutationrepresents a single amino acid deletion, insertion or substitution. 11.A purified or recombinant polypeptide comprising an amino acid of SEQ IDNO:
 9. 12. A nucleic acid sequence comprising the sequence of SEQ ID NO:8.
 13. A transgenic host cell comprising the nucleotide sequence ofclaim
 12. 14. A nucleic acid sequence comprising a 100 bp nucleic acidsequence that is identical to a contiguous 100 bp sequence of SEQ ID NO:8.
 15. A method of screening for potential human therapeutic agents,said method comprising contacting a SAMP32 polypeptide with a candidatecompound; and determining if the candidate compound selectively binds tothe SAMP32 polypeptide.
 16. The method of claim 15 wherein the SAMP32polypeptide is expressed on the surface of a cell.
 17. An antibody thatbinds specifically to the protein of SEQ ID NO:
 9. 18. An antigeniccomposition comprising a SAMP32 polypeptide and a pharmaceuticallyacceptable carrier.
 19. A purified polypeptide comprising the amino acidsequence of SEQ ID NO: 16; an amino acid sequence that differs from SEQID NO: 16 by one or more conservative amino acid substitutions; or anamino acid sequence that differs from SEQ ID NO: 16 by a singlemutation, wherein the single mutation represents a single amino aciddeletion, insertion or substitution.
 20. A purified or recombinantpolypeptide wherein said polypeptide comprises an amino acid sequence ofSEQ ID NO:
 16. 21. A nucleic acid sequence comprising a sequenceselected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 andSEQ ID NO:
 18. 22. The nucleic acid sequence of claim 21 comprising theSEQ ID NO:
 18. 23. A transgenic host cell comprising the nucleotidesequence of claim
 21. 24. A nucleic acid sequence comprising a 100 bpnucleic acid sequence that is identical to a contiguous 100 bp sequenceof SEQ ID NO:
 14. 25. A method of screening for potential humantherapeutic agents, said method comprising contacting a C58 polypeptidewith a candidate compound; and determining if the candidate compoundselectively binds to the C58 polypeptide.
 26. The method of claim 25wherein the C58 polypeptide is expressed on the surface of a cell. 27.An antibody that binds specifically to the protein of SEQ ID NO:
 16. 28.The antibody of claim 27 wherein the antibody binds to amino acids22-112 of SEQ ID NO:
 16. 29. An antigenic composition comprising a C58polypeptide and a pharmaceutically acceptable carrier.